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1
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Goldar S, Gachumi G, Siciliano SD, Hogan NS. The role of efflux transporters in cytotoxicity and intracellular concentration of chlorpyrifos and chlorpyrifos oxon in human cell lines. Toxicol In Vitro 2024; 101:105942. [PMID: 39284535 DOI: 10.1016/j.tiv.2024.105942] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Revised: 09/07/2024] [Accepted: 09/13/2024] [Indexed: 09/21/2024]
Abstract
In this study, we investigated the role of two efflux transporters, p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), in the cytotoxicity and intracellular accumulation of the organophosphate pesticide chlorpyrifos (CPF) and its active metabolite, CPF-oxon (CPFO), in a human-derived liver cell line (HepG2) and kidney epithelial cell line (HK-2). The cytotoxicity to CPF and CPFO differed between cell lines where HK-2 had lower IC50 values which could be attributed to lower basal expression and inducibility of metabolizing enzymes, transporters, and nuclear receptors in HK-2 cells. In HepG2 cells, co-exposure of CPF with a specific inhibitor of either P-gp or BCRP enhanced the cytotoxicity of CPF while co-exposure of CPFO with VRP enhanced the cytotoxicity of CPFO, suggesting the role of these transporters in the elimination CPF and CPFO. Inhibition of efflux transporters did not affect the cytotoxicity of CPF and CPFO in HK-2 cells. Co-incubation of CPF with P-gp and BCRP inhibitors increased the intracellular concentration of CPF in HepG2 cells suggesting that both transporters play a role in limiting the cellular accumulation of CPF in HepG2 cells. Our results provide evidence that inhibition of efflux transporters can enhance CPF-induced toxicity through enhanced cellular accumulation and raises additional questions regarding how pesticide-transporter interactions may influence toxicity of mixtures containing pesticides and other environmental chemicals.
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Affiliation(s)
- Samira Goldar
- Toxicology Graduate Program, Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
| | - George Gachumi
- Department of Soil Science, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada
| | - Steven D Siciliano
- Department of Soil Science, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada; Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
| | - Natacha S Hogan
- Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada; Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada.
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2
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Davies KA, Welch SR, Sorvillo TE, Coleman-McCray JD, Martin ML, Brignone JM, Montgomery JM, Spiropoulou CF, Spengler JR. Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses. Sci Rep 2023; 13:19384. [PMID: 37938597 PMCID: PMC10632498 DOI: 10.1038/s41598-023-45740-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2023] [Accepted: 10/23/2023] [Indexed: 11/09/2023] Open
Abstract
Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean-Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species (Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility.
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Affiliation(s)
- Katherine A Davies
- Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA
- U.S. Department of Agriculture, Agricultural Research Service, Zoonotic and Emerging Disease Research Unit, National Bio and Agro-Defense Facility, Manhattan, KS, USA
| | - Stephen R Welch
- Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Teresa E Sorvillo
- Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - JoAnn D Coleman-McCray
- Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - María Laura Martin
- Departamento Investigación, Instituto Nacional de Enfermedades Virales Humanas (INEVH) "Dr. Julio I. Maiztegui", Pergamino, Argentina
| | - Julia M Brignone
- Departamento Investigación, Instituto Nacional de Enfermedades Virales Humanas (INEVH) "Dr. Julio I. Maiztegui", Pergamino, Argentina
| | - Joel M Montgomery
- Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Christina F Spiropoulou
- Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Jessica R Spengler
- Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA.
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3
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Hu Y, Jiang Q, Zhai X, Liu L, Hong Y. Screening and validation of the optimal panel of reference genes in colonic epithelium and relative cancer cell lines. Sci Rep 2023; 13:17777. [PMID: 37853035 PMCID: PMC10584832 DOI: 10.1038/s41598-023-45174-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Accepted: 10/17/2023] [Indexed: 10/20/2023] Open
Abstract
Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common method to determine mRNA expression, and Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that a panel of reference genes for RT-qPCR is conducive to obtaining accurate results. This study aimed to screen and verify the optimal panel of reference genes in colorectal cancer (CRC) and normal colonic cell lines. In the study, eight candidate reference genes (GAPDH, ACTB, 18S, PPIA, B2M, SDHA, GUSB, and YWHAZ) were selected for RT-qPCR to detect their expression in NCM460, HT29, HCT116, SW480, SW620, DLD-1, LOVO and RKO cell lines. The stability of reference genes and the optimal panel were evaluated by geNorm, NormFinder, and BestKeeper software. As results, the expression levels of candidate reference genes differed in the colonic epithelial cell lines, and the number of optimal panel of reference genes is two. B2M and YWHAZ were the two most stable reference genes for NCM460, HCT116, SW620, LOVO, and RKO cell lines, while only one of B2M and YWHAZ was most stable in HT29 and SW480 cells. In DLD-1 cells, the stability of B2M and YWHAZ ranked 3rd and 6th, PPIA and GUSB were the most stable two. Furthermore, the YWHZA + B2M performed smaller intragroup differences than other panel or single reference gene. In conclusion, this study indicates the optimal panel of reference genes is YWHZA + B2M for the NCM460, HCT116, SW620, LOVO, RKO, SW480, and HT29 cell lines, but it is PPIA + GUSB in DLD-1 cell lines.
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Affiliation(s)
- Yang Hu
- Department of Gastroenterology, The First People's Hospital of Jiande, Hangzhou, 311600, China
| | - Qi Jiang
- Department of Biological Repositories, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
| | - Xiang Zhai
- Key Laboratory of Intestinal and Colorectal Diseases of Hubei Province, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
| | - Liang Liu
- Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
| | - Yuntian Hong
- Key Laboratory of Intestinal and Colorectal Diseases of Hubei Province, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
- Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
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4
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Selection of suitable reference gene for gene expression studies of porcine ovaries under different conditions in quantitative reverse transcription polymerase chain reaction assay. JOURNAL OF ANIMAL REPRODUCTION AND BIOTECHNOLOGY 2022. [DOI: 10.12750/jarb.37.2.96] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
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5
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Fan H, He Q, Dong Y, Xu W, Lou Y, Hua X, Xu T. Selection of suitable candidate genes for mRNA expression normalization in bulbil development of Pinellia ternata. Sci Rep 2022; 12:8849. [PMID: 35614175 PMCID: PMC9133075 DOI: 10.1038/s41598-022-12782-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2022] [Accepted: 05/03/2022] [Indexed: 11/09/2022] Open
Abstract
Pinellia ternata (Thunb.) Breit. (Abbreviated as P. ternata). It is a commonly prescribed Chinese traditional medicinal herb for the treatment of phlegm, cough, and morning sick. Bulbil reproduction is one of the main reproductive methods of P. ternata. The accurate quantification of gene expression patterns associated with bulbil development might be helpful to explore the molecular mechanism involved in P. ternata reproduction. Quantitative real-time PCR was the most preferred method for expression profile and function analysis of mRNA. However, the reference genes in different tissues of P. ternata in different periods of bulbil development have not been studied in detail. In present study, the expression stability of eight candidate reference genes were determined with programs: geNorm, NormFinder, BestKeeper, and refFinder. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the top- rated reference gene in all samples of P. ternata, while different combinations of reference gene proved to be the most stable depending on development stage and tissue type. Furthermore, the reliability of GAPDH expression was verified by six P. ternata related genes in hormone and nutrient biosynthesis pathways, and the expression profiles of these genes were agreed with the results of RNA-seq digital gene expression analysis. These results can contribute to studies of gene expression patterns and functional analysis of P. ternata involved in bulbil development.
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Affiliation(s)
- Haoyu Fan
- Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou, China
| | - Qiuling He
- Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou, China.
| | - Yiheng Dong
- Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou, China
| | - Wenxin Xu
- Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou, China
| | - Yanlin Lou
- Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou, China
| | - Xuejun Hua
- Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou, China
| | - Tao Xu
- Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou, China.
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Cadenas J, Pors SE, Nikiforov D, Zheng M, Subiran C, Bøtkjær JA, Mamsen LS, Kristensen SG, Andersen CY. Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue. Int J Mol Sci 2022; 23:ijms23020886. [PMID: 35055072 PMCID: PMC8778884 DOI: 10.3390/ijms23020886] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Revised: 01/08/2022] [Accepted: 01/11/2022] [Indexed: 11/30/2022] Open
Abstract
Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral follicles, ovarian medulla, and ovarian cortex tissue. The selected genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), large ribosomal protein P0 (RPLP0), beta-actin (ACTB), and peptidylprolyl isomerase A (PPIA). Overall, the stability of all RGs differed among ovarian cell types and tissues. NormFinder identified ACTB as the best RG for oocytes and cumulus cells, and B2M for medulla tissue and isolated follicles. The combination of two RGs only marginally increased the stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For the ovarian cortex, depending on culture conditions, GAPDH or ACTB were found to be the most stable genes. Our results highlight the importance of assessing RGs for each cell type or tissue when performing RT-qPCR analysis.
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da Conceição Braga L, Gonçalves BÔP, Coelho PL, da Silva Filho AL, Silva LM. Identification of best housekeeping genes for the normalization of RT-qPCR in human cell lines. Acta Histochem 2022; 124:151821. [PMID: 34861601 DOI: 10.1016/j.acthis.2021.151821] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2021] [Revised: 11/22/2021] [Accepted: 11/25/2021] [Indexed: 11/01/2022]
Abstract
The identification of the best reference gene is a critical step to evaluate the relative change in mRNA expression of a target gene by RT-qPCR. In this work, we evaluated nineteen genes of different functional classes using Real Time Human Reference Gene Panel (Roche Applied Sciences), to identify the internal housekeeping genes (HKGs) most suitable for gene expression normalization data in human cell lines. Normal cell lines CCD-19LU (lung fibroblast), HEK-293 (epithelial cell of embryonic kidney), WI-26 VA4 (lung fibroblast), and human cancer cells, BT-549 (breast cancer), Hs 578T (breast cancer), MACL-1 (breast cancer), HeLa (cervical carcinoma), U-87 MG (glioblastoma/astrocytoma), RKO-AS45-1 (colorectal carcinoma), and TOV-21G (ovarian adenocarcinoma) were cultivated according to manufacturer's protocol. Twelve candidate reference genes were commonly expressed in five cell lines (CCD-19Lu, HEK-293, RKO-AS45-1, TOV-21G, and U-87 MG). To verify the expression stability, we used the RefFinder web tool, which integrates data from the computational programs Normfinder, BestKeeper, geNorm, and the comparative Delta-Ct method. The ACTB was the most stable reference gene to the CCD-19Lu and HEK-293 cells. The best combination of HKGs for the RKO-AS45-1 and TOV-21G cell lines were B2M/GAPDH and PBGD/B2M, respectively. For the U-87 MG cells, GAPDH and IPO8 were the most suitable HKGs. Thus, our findings showed that it is crucial to use the right HKGs to precise normalize gene expression levels in cancer studies, once a suitable HKG for one cell type cannot be to the other.
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8
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Eskandari R, Asoodeh A, Mousavi SD, Firouzi Z. The effect of a novel drug delivery system using encapsulated antimicrobial peptide Protonectin (IL-12) into Nano micelle PEG-PCL on A549 adenocarcinoma lung cell line. JOURNAL OF POLYMER RESEARCH 2021. [DOI: 10.1007/s10965-021-02699-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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9
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Iness AN, Rubinsak L, Meas SJ, Chaoul J, Sayeed S, Pillappa R, Temkin SM, Dozmorov MG, Litovchick L. Oncogenic B-Myb Is Associated With Deregulation of the DREAM-Mediated Cell Cycle Gene Expression Program in High Grade Serous Ovarian Carcinoma Clinical Tumor Samples. Front Oncol 2021; 11:637193. [PMID: 33747961 PMCID: PMC7969987 DOI: 10.3389/fonc.2021.637193] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Accepted: 02/08/2021] [Indexed: 12/21/2022] Open
Abstract
Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.
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Affiliation(s)
- Audra N Iness
- Division of Hematology, Oncology and Palliative Care, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, United States
| | - Lisa Rubinsak
- Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA, United States
| | - Steven J Meas
- School of Medicine, Virginia Commonwealth University, Richmond, VA, United States
| | - Jessica Chaoul
- School of Medicine, Virginia Commonwealth University, Richmond, VA, United States
| | - Sadia Sayeed
- Department of Pathology, Virginia Commonwealth University, Richmond, VA, United States.,Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, United States
| | - Raghavendra Pillappa
- Department of Pathology, Virginia Commonwealth University, Richmond, VA, United States
| | - Sarah M Temkin
- Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA, United States
| | - Mikhail G Dozmorov
- Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, United States.,Department of Biostatistics, Virginia Commonwealth University, Richmond, VA, United States.,Department of Pathology, Virginia Commonwealth University, Richmond, VA, United States
| | - Larisa Litovchick
- Division of Hematology, Oncology and Palliative Care, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, United States.,Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, United States
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10
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Eskandari R, Asoodeh A, Behnam-Rassouli F. The Therapeutic Effects of an Antimicrobial Peptide Protonectin (IL-12) on A549 Cancer Cell Line. Int J Pept Res Ther 2020. [DOI: 10.1007/s10989-020-10116-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Asiabi P, Ambroise J, Giachini C, Coccia ME, Bearzatto B, Chiti MC, Dolmans MM, Amorim CA. Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries. J Assist Reprod Genet 2020; 37:2545-2553. [PMID: 32729067 DOI: 10.1007/s10815-020-01901-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Accepted: 07/23/2020] [Indexed: 12/13/2022] Open
Abstract
PURPOSE Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes. METHODS RNA was isolated from 5 normal human ovaries (52-79 years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49-75 years of age), and 4 PCOS ovaries (18-35 years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate. RESULTS Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes. CONCLUSIONS This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group.
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Affiliation(s)
- P Asiabi
- Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte B1.52.02, 1200, Brussels, Belgium
| | - J Ambroise
- Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, 1200, Brussels, Belgium
| | - C Giachini
- Department of Clinical and Experimental Biomedical Sciences, University of Florence, 50134, Florence, Italy
| | - M E Coccia
- Department of Clinical and Experimental Biomedical Sciences, University of Florence, 50134, Florence, Italy
| | - B Bearzatto
- Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, 1200, Brussels, Belgium
| | - M C Chiti
- Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte B1.52.02, 1200, Brussels, Belgium
| | - M M Dolmans
- Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte B1.52.02, 1200, Brussels, Belgium
- Gynecology Department, Cliniques Universitaires Saint-Luc, 1200, Brussels, Belgium
| | - C A Amorim
- Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte B1.52.02, 1200, Brussels, Belgium.
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Liu P, Zhong Y, Cao T, Sheng X, Huang H. A frequent somatic mutation in the 3'UTR of GAPDH facilitates the development of ovarian cancer by creating a miR‑125b binding site. Oncol Rep 2020; 44:887-896. [PMID: 32705257 PMCID: PMC7388293 DOI: 10.3892/or.2020.7663] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Accepted: 04/23/2020] [Indexed: 12/27/2022] Open
Abstract
Ovarian cancer (OVCA) is one of the most common types of cancer in women worldwide. Recent studies have focused on the presence and effect of somatic mutations in patients with OVCA; however, studies on the roles of mutations located in the untranslated regions (UTR) of genes in OVCA remain limited. In the present study, a frequent somatic mutation in the glyceraldehyde 3-phosphate dehydrogenase (GADPH) 3′UTR was identified using transcriptome sequencing of 120 pairs of OVCA tissue samples. The mutant GAPDH 3′UTR promoted tumor growth and cell motility. Furthermore, the mutation in the GAPDH 3′UTR significantly downregulated the levels of mature miR-125b by creating a new miR-125b binding site. Finally, STAT3 levels were increased in SKOV3 cells stably expressing the mutant GADPH 3′UTR, which is a critical target gene of miR-125b. In conclusion, the present study demonstrated that the mutation located in GAPDH 3′UTR promoted OVCA growth and development by sponging miR-125b and thereby affecting STAT3 expression levels.
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Affiliation(s)
- Peisen Liu
- Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510150, P.R. China
| | - Yumin Zhong
- Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510150, P.R. China
| | - Ting Cao
- Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510150, P.R. China
| | - Xiujie Sheng
- Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510150, P.R. China
| | - Huang Huang
- Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510150, P.R. China
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13
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Hsu YH, Wang PH, Chang CM. Functional Gene Clusters in Global Pathogenesis of Clear Cell Carcinoma of the Ovary Discovered by Integrated Analysis of Transcriptomes. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 2020; 17:ijerph17113951. [PMID: 32498447 PMCID: PMC7312065 DOI: 10.3390/ijerph17113951] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/27/2020] [Revised: 05/23/2020] [Accepted: 05/31/2020] [Indexed: 12/17/2022]
Abstract
Clear cell carcinoma of the ovary (ovarian clear cell carcinoma (OCCC)) is one epithelial ovarian carcinoma that is known to have a poor prognosis and a tendency for being refractory to treatment due to unclear pathogenesis. Published investigations of OCCC have mainly focused only on individual genes and lack of systematic integrated research to analyze the pathogenesis of OCCC in a genome-wide perspective. Thus, we conducted an integrated analysis using transcriptome datasets from a public domain database to determine genes that may be implicated in the pathogenesis involved in OCCC carcinogenesis. We used the data obtained from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) DataSets. We found six interactive functional gene clusters in the pathogenesis network of OCCC, including ribosomal protein, eukaryotic translation initiation factors, lactate, prostaglandin, proteasome, and insulin-like growth factor. This finding from our integrated analysis affords us a global understanding of the interactive network of OCCC pathogenesis.
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Affiliation(s)
- Yueh-Han Hsu
- Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, Taipei 112, Taiwan; (Y.-H.H.); (P.-H.W.)
- School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
| | - Peng-Hui Wang
- Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, Taipei 112, Taiwan; (Y.-H.H.); (P.-H.W.)
- School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
- Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan
- Department of Medical Research, China Medical University Hospital, Taichung 440, Taiwan
- Female Cancer Foundation, Taipei 104, Taiwan
| | - Chia-Ming Chang
- Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, Taipei 112, Taiwan; (Y.-H.H.); (P.-H.W.)
- School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
- Correspondence: ; Tel.: +886-2-2875-7826; Fax: +886-2-5570-2788
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14
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Clark KL, Keating AF. Ataxia-telangiectasia mutated coordinates the ovarian DNA repair and atresia-initiating response to phosphoramide mustard. Biol Reprod 2020; 102:248-260. [PMID: 31435664 DOI: 10.1093/biolre/ioz160] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2019] [Revised: 07/31/2019] [Accepted: 08/13/2019] [Indexed: 11/13/2022] Open
Abstract
Ataxia-telangiectasia-mutated (ATM) protein recognizes and repairs DNA double strand breaks through activation of cell cycle checkpoints and DNA repair proteins. Atm gene mutations increase female reproductive cancer risk. Phosphoramide mustard (PM) induces ovarian DNA damage and destroys primordial follicles, and pharmacological ATM inhibition prevents PM-induced follicular depletion. Wild-type (WT) C57BL/6 or Atm+/- mice were dosed once intraperitoneally with sesame oil (95%) or PM (25 mg/kg) in the proestrus phase of the estrous cycle and ovaries harvested 3 days thereafter. Atm+/- mice spent ~25% more time in diestrus phase than WT. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) on ovarian protein was performed and bioinformatically analyzed. Relative to WT, Atm+/- mice had 64 and 243 proteins increased or decreased in abundance, respectively. In WT mice, PM increased 162 and decreased 20 proteins. In Atm+/- mice, 173 and 37 proteins were increased and decreased, respectively, by PM. Exportin-2 (XPO2) was localized to granulosa cells of all follicle stages and was 7.2-fold greater in Atm+/- than WT mice. Cytoplasmic FMR1-interacting protein 1 was 6.8-fold lower in Atm+/- mice and was located in the surface epithelium with apparent translocation to the ovarian medulla post-PM exposure. PM induced γH2AX, but fewer γH2AX-positive foci were identified in Atm+/- ovaries. Similarly, cleaved caspase-3 was lower in the Atm+/- PM-treated, relative to WT mice. These findings support ATM involvement in ovarian DNA repair and suggest that ATM functions to regulate ovarian atresia.
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Affiliation(s)
- Kendra L Clark
- Department of Animal Science, Iowa State University, Ames, Iowa 50011, USA
| | - Aileen F Keating
- Department of Animal Science, Iowa State University, Ames, Iowa 50011, USA
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15
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Kloubert V, Rink L. Selection of an inadequate housekeeping gene leads to misinterpretation of target gene expression in zinc deficiency and zinc supplementation models. J Trace Elem Med Biol 2019; 56:192-197. [PMID: 31513994 DOI: 10.1016/j.jtemb.2019.08.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/22/2019] [Revised: 08/19/2019] [Accepted: 08/19/2019] [Indexed: 01/21/2023]
Abstract
BACKGROUND Numerous studies try to find the most stable housekeeping gene for a specific experimental setup. However, there is no universal housekeeping gene described so far and, therefore, new testing of housekeeping genes at the beginning of a new experiment is of high importance. METHODS In the present study, target gene expression of mitochondrial serine/threonine-protein phosphatase (PGAM)5, nuclear factor erythroid 2-related factor (Nrf)2, dynamin related protein (Drp)1 and kelch like ECH associated protein (Keap)1 was tested in zinc-deficient and zinc-supplemented THP-1 cells and compared to control cells. Normalization of results obtained by quantitative real-time polymerase chain reaction (qRT-PCR) was performed by using the housekeeping genes porphobilinogen deaminase (PBGD), β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Additionally, this 3 housekeeping genes were tested in Jurkat cells under these conditions. RESULTS Surprisingly, analyses of one and the same target gene revealed opposite results depending on the used housekeeping gene. This was caused by significant altered housekeeping gene expressions due to zinc availability. CONCLUSION Therefore, this study highlights the importance of choosing adequate housekeeping genes, which might comprise the use of more than one housekeeping gene when different conditions are tested, such as zinc deficiency and zinc supplementation. Related to the herein used experimental setup, the use ofGAPDH to study gene expression upon zinc deficiency and PBGD to study gene expression after zinc supplementation is recommended.
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Affiliation(s)
- Veronika Kloubert
- Institute of Immunology, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany.
| | - Lothar Rink
- Institute of Immunology, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany.
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16
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Wright Muelas M, Mughal F, O'Hagan S, Day PJ, Kell DB. The role and robustness of the Gini coefficient as an unbiased tool for the selection of Gini genes for normalising expression profiling data. Sci Rep 2019; 9:17960. [PMID: 31784565 PMCID: PMC6884504 DOI: 10.1038/s41598-019-54288-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2019] [Accepted: 11/08/2019] [Indexed: 12/13/2022] Open
Abstract
We recently introduced the Gini coefficient (GC) for assessing the expression variation of a particular gene in a dataset, as a means of selecting improved reference genes over the cohort ('housekeeping genes') typically used for normalisation in expression profiling studies. Those genes (transcripts) that we determined to be useable as reference genes differed greatly from previous suggestions based on hypothesis-driven approaches. A limitation of this initial study is that a single (albeit large) dataset was employed for both tissues and cell lines. We here extend this analysis to encompass seven other large datasets. Although their absolute values differ a little, the Gini values and median expression levels of the various genes are well correlated with each other between the various cell line datasets, implying that our original choice of the more ubiquitously expressed low-Gini-coefficient genes was indeed sound. In tissues, the Gini values and median expression levels of genes showed a greater variation, with the GC of genes changing with the number and types of tissues in the data sets. In all data sets, regardless of whether this was derived from tissues or cell lines, we also show that the GC is a robust measure of gene expression stability. Using the GC as a measure of expression stability we illustrate its utility to find tissue- and cell line-optimised housekeeping genes without any prior bias, that again include only a small number of previously reported housekeeping genes. We also independently confirmed this experimentally using RT-qPCR with 40 candidate GC genes in a panel of 10 cell lines. These were termed the Gini Genes. In many cases, the variation in the expression levels of classical reference genes is really quite huge (e.g. 44 fold for GAPDH in one data set), suggesting that the cure (of using them as normalising genes) may in some cases be worse than the disease (of not doing so). We recommend the present data-driven approach for the selection of reference genes by using the easy-to-calculate and robust GC.
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Affiliation(s)
- Marina Wright Muelas
- Department of Biochemistry, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK.
| | - Farah Mughal
- Department of Biochemistry, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK
| | - Steve O'Hagan
- School of Chemistry, Department of Chemistry, The Manchester Institute of Biotechnology 131, Princess Street, Manchester, M1 7DN, UK
- The Manchester Institute of Biotechnology, 131, Princess Street, Manchester, M1 7DN, UK
| | - Philip J Day
- The Manchester Institute of Biotechnology, 131, Princess Street, Manchester, M1 7DN, UK.
- Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, M13 9PL, UK.
| | - Douglas B Kell
- Department of Biochemistry, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK.
- Novo Nordisk Foundation Centre for Biosustainability, Technical University of Denmark, 10 Building 220, Kemitorvet, 2800, Kgs. Lyngby, Denmark.
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17
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Validation of Reference Genes for Normalization of Relative qRT-PCR Studies in Papillary Thyroid Carcinoma. Sci Rep 2019; 9:15241. [PMID: 31645594 PMCID: PMC6811563 DOI: 10.1038/s41598-019-49247-1] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Accepted: 08/19/2019] [Indexed: 12/21/2022] Open
Abstract
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) in thyroid tumors require accurate data normalization, however, there are no sufficient studies addressing the suitable reference genes for gene expression analysis in malignant and normal thyroid tissue specimens. The purpose of this study was to identify valid internal control genes for normalization of relative qRT-PCR studies in human papillary thyroid carcinoma tissue samples. The expression characteristics of 12 candidate reference genes (GAPDH, ACTB, HPRT1, TBP, B2M, PPIA, 18SrRNA, HMBS, GUSB, PGK1, RPLP0, and PGM1) were assessed by qRT-PCR in 45 thyroid tissue samples (15 papillary thyroid carcinoma, 15 paired normal tissues and 15 multinodular goiters). These twelve candidate reference genes were selected by a systematic literature search. GeNorm, NormFinder, and BestKeeper statistical algorithms were applied to determine the most stable reference genes. The three algorithms were in agreement in identifying GUSB and HPRT1 as the most stably expressed genes in all thyroid tumors investigated. According to the NormFinder software, the pair of genes including ‘GUSB and HPRT1’ or ‘GUSB and HMBS’ or ‘GUSB and PGM1’ were the best combinations for selection of pair reference genes. The optimal number of genes required for reliable normalization of qPCR data in thyroid tissues would be three according to calculations made by GeNorm algorithm. These results suggest that GUSB and HPRT1 are promising reference genes for normalization of relative qRT-PCR studies in papillary thyroid carcinoma.
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18
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Molavi G, Samadi N, Hosseingholi EZ. The roles of moonlight ribosomal proteins in the development of human cancers. J Cell Physiol 2018; 234:8327-8341. [PMID: 30417503 DOI: 10.1002/jcp.27722] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2018] [Accepted: 09/23/2018] [Indexed: 12/13/2022]
Abstract
"Moonlighting protein" is a term used to define a single protein with multiple functions and different activities that are not derived from gene fusions, multiple RNA splicing, or the proteolytic activity of promiscuous enzymes. Different proteinous constituents of ribosomes have been shown to have important moonlighting extra-ribosomal functions. In this review, we introduce the impact of key moonlight ribosomal proteins and dependent signal transduction in the initiation and progression of various cancers. As a future perspective, the potential role of these moonlight ribosomal proteins in the diagnosis, prognosis, and development of novel strategies to improve the efficacy of therapies for human cancers has been suggested.
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Affiliation(s)
- Ghader Molavi
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.,Department of Molecular Medicine, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Nasser Samadi
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.,Department of Molecular Medicine, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.,Department of Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
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19
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O'Hagan S, Wright Muelas M, Day PJ, Lundberg E, Kell DB. GeneGini: Assessment via the Gini Coefficient of Reference "Housekeeping" Genes and Diverse Human Transporter Expression Profiles. Cell Syst 2018; 6:230-244.e1. [PMID: 29428416 PMCID: PMC5840522 DOI: 10.1016/j.cels.2018.01.003] [Citation(s) in RCA: 49] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2017] [Revised: 09/26/2017] [Accepted: 12/30/2017] [Indexed: 01/13/2023]
Abstract
The expression levels of SLC or ABC membrane transporter transcripts typically differ 100- to 10,000-fold between different tissues. The Gini coefficient characterizes such inequalities and here is used to describe the distribution of the expression of each transporter among different human tissues and cell lines. Many transporters exhibit extremely high Gini coefficients even for common substrates, indicating considerable specialization consistent with divergent evolution. The expression profiles of SLC transporters in different cell lines behave similarly, although Gini coefficients for ABC transporters tend to be larger in cell lines than in tissues, implying selection. Transporter genes are significantly more heterogeneously expressed than the members of most non-transporter gene classes. Transcripts with the stablest expression have a low Gini index and often differ significantly from the "housekeeping" genes commonly used for normalization in transcriptomics/qPCR studies. PCBP1 has a low Gini coefficient, is reasonably expressed, and is an excellent novel reference gene. The approach, referred to as GeneGini, provides rapid and simple characterization of expression-profile distributions and improved normalization of genome-wide expression-profiling data.
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Affiliation(s)
- Steve O'Hagan
- School of Chemistry, 131, Princess Street, Manchester M1 7DN, UK; The Manchester Institute of Biotechnology, 131, Princess Street, Manchester M1 7DN, UK
| | - Marina Wright Muelas
- School of Chemistry, 131, Princess Street, Manchester M1 7DN, UK; The Manchester Institute of Biotechnology, 131, Princess Street, Manchester M1 7DN, UK
| | - Philip J Day
- The Manchester Institute of Biotechnology, 131, Princess Street, Manchester M1 7DN, UK; Faculty of Biology, Medicine and Health, The University of Manchester, Oxford Road, Manchester M13 9PL, UK
| | - Emma Lundberg
- Science for Life Laboratory, Royal Institute of Technology (KTH), SE-17121 Solna, Sweden.
| | - Douglas B Kell
- School of Chemistry, 131, Princess Street, Manchester M1 7DN, UK; The Manchester Institute of Biotechnology, 131, Princess Street, Manchester M1 7DN, UK.
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20
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PUM1 promotes ovarian cancer proliferation, migration and invasion. Biochem Biophys Res Commun 2018; 497:313-318. [DOI: 10.1016/j.bbrc.2018.02.078] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Accepted: 02/07/2018] [Indexed: 01/30/2023]
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21
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Zhao H, Xu H, Xue L. Regulatory network involving miRNAs and genes in serous ovarian carcinoma. Oncol Lett 2017; 14:6259-6268. [PMID: 29113276 DOI: 10.3892/ol.2017.6927] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2015] [Accepted: 05/23/2017] [Indexed: 12/19/2022] Open
Abstract
Serous ovarian carcinoma (SOC) is one of the most life-threatening types of gynecological malignancy, but the pathogenesis of SOC remains unknown. Previous studies have indicated that differentially expressed genes and microRNAs (miRNAs) serve important functions in SOC. However, genes and miRNAs are identified in a disperse form, and limited information is known about the regulatory association between miRNAs and genes in SOC. In the present study, three regulatory networks were hierarchically constructed, including a differentially-expressed network, a related network and a global network to reveal associations between each factor. In each network, there were three types of factors, which were genes, miRNAs and transcription factors that interact with each other. Focus was placed on the differentially-expressed network, in which all genes and miRNAs were differentially expressed and therefore may have affected the development of SOC. Following the comparison and analysis between the three networks, a number of signaling pathways which demonstrated differentially expressed elements were highlighted. Subsequently, the upstream and downstream elements of differentially expressed miRNAs and genes were listed, and a number of key elements (differentially expressed miRNAs, genes and TFs predicted using the P-match method) were analyzed. The differentially expressed network partially illuminated the pathogenesis of SOC. It was hypothesized that if there was no differential expression of miRNAs and genes, SOC may be prevented and treatment may be identified. The present study provided a theoretical foundation for gene therapy for SOC.
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Affiliation(s)
- Haiyan Zhao
- College of Computer Science and Technology, Jilin University, Changchun, Jilin 130012, P.R. China
| | - Hao Xu
- College of Computer Science and Technology, Jilin University, Changchun, Jilin 130012, P.R. China.,Zhuhai Laboratory of Key Laboratory of Symbol Computation and Knowledge Engineering of Ministry of Education, Department of Computer Science and Technology, Zhuhai College of Jilin University, Zhuhai, Guangdong 519041, P.R. China
| | - Luchen Xue
- College of Software, Jilin University, Changchun, Jilin 130012, P.R. China
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22
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Identification of TMEM208 and PQLC2 as reference genes for normalizing mRNA expression in colorectal cancer treated with aspirin. Oncotarget 2017; 8:22759-22771. [PMID: 28184026 PMCID: PMC5410260 DOI: 10.18632/oncotarget.15191] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2016] [Accepted: 01/23/2017] [Indexed: 12/21/2022] Open
Abstract
Numerous evidences indicate that aspirin usage causes a significant reduction in colorectal cancer. However, the molecular mechanisms about aspirin preventing colon cancer are largely unknown. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a most frequently used method to identify the target molecules regulated by certain compound. However, this method needs stable internal reference genes to analyze the expression change of the targets. In this study, the transcriptional stabilities of several traditional reference genes were evaluated in colon cancer cells treated with aspirin, and also, the suitable internal reference genes were screened by using a microarray and were further identified by using the geNorm and NormFinder softwares, and then were validated in more cell lines and xenografts. We have showed that three traditional internal reference genes, β-actin, GAPDH and α-tubulin, are not suitable for studying gene transcription in colon cancer cells treated with aspirin, and we have identified and validated TMEM208 and PQLC2 as the ideal internal reference genes for detecting the molecular targets of aspirin in colon cancer in vitro and in vivo. This study reveals stable internal reference genes for studying the target genes of aspirin in colon cancer, which will contribute to identify the molecular mechanism behind aspirin preventing colon cancer.
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23
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Zhang C, Wang YQ, Jin G, Wu S, Cui J, Wang RF. Selection of reference genes for gene expression studies in human bladder cancer using SYBR-Green quantitative polymerase chain reaction. Oncol Lett 2017; 14:6001-6011. [PMID: 29113238 PMCID: PMC5661485 DOI: 10.3892/ol.2017.7002] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2016] [Accepted: 07/20/2017] [Indexed: 01/30/2023] Open
Abstract
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a rapid, reliable and widely used method of studying gene expression profiles that requires appropriate normalization for accurate and reliable results. Reference genes are usually used to normalize mRNA levels; however, the expression levels of these reference genes may vary between cell types, developmental stages, species and experimental conditions. Therefore, a normalization strategy is an important precondition for reliable conclusions, with endogenous controls requiring determination for every experimental system. In the present study, 18 reference genes used in various prior studies were analyzed to determine their applicability in bladder cancer. A total of 35 matched malignant and non-malignant bladder cancer (specifically transitional cell carcinoma) tissue specimens were examined. RNA and cDNA quality was stringently controlled. Candidate reference genes were assessed using SYBR-Green RT-qPCR. mRNA abundance was compared and reference genes with distinct ranges of expression to possible target genes were excluded. Genes that were differentially expressed in matched non-cancerous and cancerous samples were also excluded, using quantification cycle analysis. Subsequently, the stability of the selected reference genes was analyzed using three different methods: geNorm, NormFinder and BestKeeper. The rarely used ribosomal protein S23 (RPS23) was the most stable single reference gene, with RPS23, tumor protein, translationally controlled 1 and RPS13 comprising the optimal reference gene set for all the bladder samples. These stable reference genes should be employed in normalization and quantification of transcript levels in future expression studies of bladder cancer-associated genes.
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Affiliation(s)
- Chuanxia Zhang
- Key Laboratory of Gene Engineering of The Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510275, P.R. China.,Zhongshan School of Medicine, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, State Key Laboratory of Oncology in South China, Guangzhou, Guangdong 510060, P.R. China
| | - Yong Qiang Wang
- Department of Urology, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, State Key Laboratory of Oncology in South China, Guangzhou, Guangdong 510060, P.R. China
| | - Guangyi Jin
- Shenzhen Key Laboratory of Translational Medicine of Tumor, Cancer Research Center, School of Medicine, Shenzhen University, Shenzhen, Guangdong 518060, P.R. China
| | - Song Wu
- Department of Urological Surgery, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Guangzhou, Guangdong 510275, P.R. China
| | - Jun Cui
- Key Laboratory of Gene Engineering of The Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510275, P.R. China.,Collaborative Innovation Center of Cancer Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510275, P.R. China
| | - Rong-Fu Wang
- Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX 77030, USA.,Department of Microbiology and Immunology, Weill Cornell Medical College, Cornell University, New York, NY 10065, USA.,Institute of Biosciences and Technology, College of Medicine, Texas A & M University, Houston, TX 77030, USA
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24
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Neuropilin-1 Associated Molecules in the Blood Distinguish Poor Prognosis Breast Cancer: A Cross-Sectional Study. Sci Rep 2017; 7:3301. [PMID: 28607365 PMCID: PMC5468252 DOI: 10.1038/s41598-017-03280-0] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2017] [Accepted: 04/28/2017] [Indexed: 12/12/2022] Open
Abstract
Circulating plasma and peripheral blood mononuclear (PBMCs) cells provide an informative snapshot of the systemic physiological state. Moreover, they provide a non-invasively accessible compartment to identify biomarkers for personalized medicine in advanced breast cancer. The role of Neuropilin-1 (NRP-1) and its interacting molecules in breast tumor tissue was correlated with cancer progression; however, the clinical impact of their systemic levels was not extensively evaluated. In this cross-sectional study, we found that circulating and tumor tissue expression of NRP-1 and circulating placental growth factor (PlGF) increase in advanced nodal and metastatic breast cancer compared with locally advanced disease. Tumor tissue expression of NRP-1 and PlGF is also upregulated in triple negative breast cancer (TNBC) compared to other subtypes. Conversely, in PBMCs, NRP-1 and its interacting molecules SEMA4A and SNAI1 are significantly downregulated in breast cancer patients compared to healthy controls, indicating a protective role. Moreover, we report differential PBMC expression profiles that correlate inversely with disease stage (SEMA4A, SNAI1, PLXNA1 and VEGFR3) and can differentiate between the TNBC and non-TNBC tumor subtypes (VEGFR3 and PLXNA1). This work supports the importance of NRP-1-associated molecules in circulation to characterize poor prognosis breast cancer and emphasizes on their role as favorable drug targets.
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25
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Liu Y, Li H, Ban Z, Nai M, Yang L, Chen Y, Xu Y. Annexin A2 inhibition suppresses ovarian cancer progression via regulating β-catenin/EMT. Oncol Rep 2017; 37:3643-3650. [PMID: 28440436 DOI: 10.3892/or.2017.5578] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2016] [Accepted: 03/16/2017] [Indexed: 01/23/2023] Open
Abstract
Annexin A2 is a member of the Annexin family that acts as a Ca2+-dependent phospholipid and membrane binding protein, which is associated with the survival and spread of multiple neoplasms. However, the function of Annexin A2 in ovarian cancer progression remains unclear. In this study, we aimed to investigate the role and underlying molecular mechanism of Annexin A2 in cell proliferation and invasion in ovarian cancer. We found that the mRNA expression of Annexin A2 was upregulated in ovarian cancer tissues and cell lines. In the loss-of-function of Annexin A2, β-catenin was indicated to be significantly suppressed and EMT constrained. Moreover, cell proliferation and invasion were both markedly inhibited by the downregulation of Annexin A2. Additionally, the overexpression of β-catenin obviously reversed the effect of Annexin A2 on EMT, and cell proliferation and invasion, indicating that Annexin A2 suppression regulated EMT through controlling β-catenin. Taken together, this study showed that Annexin A2 inhibition suppresses proliferation and invasion in ovarian cancer via β-catenin/EMT, proposing the potential role of Annexin A2 in the prevention and treatment of ovarian cancer.
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Affiliation(s)
- Yan Liu
- Department of Gynecology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China
| | - Hongyu Li
- Department of Gynecology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China
| | - Zhenying Ban
- Department of Pathology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China
| | - Manman Nai
- Department of Gynecology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China
| | - Li Yang
- Department of Gynecology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China
| | - Yannan Chen
- Department of Gynecology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China
| | - Yiming Xu
- Department of Gynecology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China
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26
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Li Y, Lu H, Ji Y, Wu S, Yang Y. Identification of genes for normalization of real-time RT-PCR data in placental tissues from intrahepatic cholestasis of pregnancy. Placenta 2016; 48:133-135. [DOI: 10.1016/j.placenta.2016.10.017] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/08/2016] [Revised: 09/22/2016] [Accepted: 10/31/2016] [Indexed: 01/16/2023]
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27
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Ofinran O, Bose U, Hay D, Abdul S, Tufatelli C, Khan R. Selection of suitable reference genes for gene expression studies in normal human ovarian tissues, borderline ovarian tumours and ovarian cancer. Mol Med Rep 2016; 14:5725-5731. [PMID: 27840988 DOI: 10.3892/mmr.2016.5933] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2016] [Accepted: 05/09/2016] [Indexed: 11/06/2022] Open
Abstract
The use of reference genes is the most common method of controlling the variation in mRNA expression during quantitative polymerase chain reaction, although the use of traditional reference genes, such as β‑actin, glyceraldehyde‑3‑phosphate dehydrogenase or 18S ribosomal RNA, without validation occasionally leads to unreliable results. Therefore, the present study aimed to evaluate a set of five commonly used reference genes to determine the most suitable for gene expression studies in normal ovarian tissues, borderline ovarian and ovarian cancer tissues. The expression stabilities of these genes were ranked using two gene stability algorithms, geNorm and NormFinder. Using geNorm, the two best reference genes in ovarian cancer were β‑glucuronidase and β‑actin. Hypoxanthine phosphoribosyltransferase‑1 and β‑glucuronidase were the most stable in ovarian borderline tumours, and hypoxanthine phosphoribosyltransferase‑1 and glyceraldehyde‑3‑phosphate dehydrogenase were the most stable in normal ovarian tissues. NormFinder ranked β‑actin the most stable in ovarian cancer, and the best combination of two genes was β‑glucuronidase and β‑actin. In borderline tumours, hypoxanthine phosphoribosyltransferase‑1 was identified as the most stable, and the best combination was hypoxanthine phosphoribosyltransferase‑1 and β‑glucuronidase. In normal ovarian tissues, β‑glucuronidase was recommended as the optimum reference gene, and the most optimum pair of reference genes was hypoxanthine phosphoribosyltransferase‑1 and β‑actin. To the best of our knowledge, this is the first study to investigate the selection of a set of reference genes for normalisation in quantitative polymerase chain reactions in different ovarian tissues, and therefore it is recommended that β‑glucuronidase, β‑actin and hypoxanthine phosphoribosyltransferase‑1 are the most suitable reference genes for such analyses.
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Affiliation(s)
- Olumide Ofinran
- Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, The University of Nottingham, Royal Derby Hospital, Derby DE22 3DT, UK
| | - Ujjal Bose
- Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, The University of Nottingham, Royal Derby Hospital, Derby DE22 3DT, UK
| | - Daniel Hay
- Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, The University of Nottingham, Royal Derby Hospital, Derby DE22 3DT, UK
| | - Summi Abdul
- Department of Gynaecological Oncology, Royal Derby Hospital, Derby DE22 3NE, UK
| | - Cristina Tufatelli
- Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, The University of Nottingham, Royal Derby Hospital, Derby DE22 3DT, UK
| | - Raheela Khan
- Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, The University of Nottingham, Royal Derby Hospital, Derby DE22 3DT, UK
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Xu X, Xiong X, Sun Y. The role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity. SCIENCE CHINA-LIFE SCIENCES 2016; 59:656-72. [DOI: 10.1007/s11427-016-0018-0] [Citation(s) in RCA: 79] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/15/2016] [Accepted: 04/06/2016] [Indexed: 01/29/2023]
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Leal MF, Arliani GG, Astur DC, Franciozi CE, Debieux P, Andreoli CV, Smith MC, Pochini ADC, Ejnisman B, Cohen M. Comprehensive selection of reference genes for expression studies in meniscus injury using quantitative real-time PCR. Gene 2016; 584:60-68. [PMID: 26968891 DOI: 10.1016/j.gene.2016.03.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2015] [Revised: 02/11/2016] [Accepted: 03/04/2016] [Indexed: 11/29/2022]
Abstract
The meniscus plays critical roles in the knee function. Meniscal tears can lead to knee osteoarthritis. Gene expression analysis may be a useful tool for understanding meniscus tears, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. We evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1 and TBP) using meniscus samples of (1) 19 patients with isolated meniscal tears, (2) 20 patients with meniscal tears and combined anterior cruciate ligament injury (ACL), and (3) 11 controls without meniscal tears. The stability of the candidate reference genes was determined using the NormFinder, geNorm, BestKeeper DataAssist and RefFinder software packages and comparative ΔCt method. Overall, HPRT1 was the best single reference gene. However, GenEx software demonstrated that two or more reference genes should be used for gene expression normalization, which was confirmed when we evaluated TGFβR1 expression using several reference gene combinations. HPRT1+TBP was the most frequently identified pair from the analysis of samples of (1) meniscal tear samples of patients with a concomitant ACL tears, (2) all meniscal tears, and (3) all samples. HPRT1+GAPDH was the most frequently identified pair from the analysis of samples of isolated meniscal tear samples and controls. In the analysis involving only controls, GAPDH+18S was the most frequently identified pair. In the analysis of only isolated meniscal tear samples and in the analysis of meniscal tear samples of patients with concomitant ACL tears and controls, both HPRT1+TBP and HPRT1+GAPDH were identified as suitable pairs. If the gene expression study aims to compare non-injured meniscus, isolated meniscal tears and meniscal tears of patients with ACL tears as three independent groups, the trio of HPRT1+TBP+GAPDH is the most suitable combination of reference genes.
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Affiliation(s)
- Mariana Ferreira Leal
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil; Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, 04023-001, São Paulo, SP, Brazil.
| | - Gustavo Gonçalves Arliani
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil
| | - Diego Costa Astur
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil
| | - Carlos Eduardo Franciozi
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil
| | - Pedro Debieux
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil
| | - Carlos Vicente Andreoli
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil
| | - Marília Cardoso Smith
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, 04023-001, São Paulo, SP, Brazil
| | - Alberto de Castro Pochini
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil
| | - Benno Ejnisman
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil
| | - Moises Cohen
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil
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Andrusiewicz M, Słowikowski B, Skibińska I, Wołuń-Cholewa M, Dera-Szymanowska A. Selection of reliable reference genes in eutopic and ectopic endometrium for quantitative expression studies. Biomed Pharmacother 2016; 78:66-73. [PMID: 26898426 DOI: 10.1016/j.biopha.2015.12.028] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2015] [Revised: 12/16/2015] [Accepted: 12/21/2015] [Indexed: 01/16/2023] Open
Abstract
PURPOSE Physiological changes during menstrual cycle cause the endometrium and endometriosis to develop specific kind of tissues, especially in regard to the gene expression profiles, which may include also housekeeping genes, commonly used as reference genes (RGs) in quantitative studies. Reverse transcription, followed by quantitative polymerase chain reaction (RT-qPCR) is the most precise and commonly used method in gene expression studies. In order to reduce effects of technical approaches and biological variability of gene's expression level, the studies often employ RGs in experimental data normalization. However, the expression of RGs is not always stable and depends on several variables. Thus, the selection of appropriate RG is one of the most significant steps to obtain reliable results in RT-qPCR-based methods. MATERIAL AND METHODS With the usage of RT-qPCR, we researched the expression of seven genes (ACTB, B2M, G6PD, GAPD, GUSB, HPRT and PPIA) as reliable reference genes in eutopic and ectopic endometrial tissue specimens obtained during standard surgery of women of reproductive age. Stability of expression level was analyzed by the most universal MS Excel plug-ins including: geNorm, NormFinder and BestKeeper. The descriptive statistics were evaluated using Statistica software. RESULTS The distribution of threshold (Ct) values was not equal. We identified genes with higher expression level (referring to Ct values) such as ACTB and B2M, medium e.g., GAPD and low expression level, e.g., G6PD and HPRT. We demonstrated that the stability of the analyzed reference genes was not homogenous, and different algorithms pointed to PPIA, GAPD and B2M as the most stable ones in eutopic and ectopic endometrium. On the contrary to these, GUSB and G6PD were the most unstable ones. CONCLUSIONS In RT-qPCR-based analyses of gene expression level in eutopic and ectopic endometrium, we strongly recommend that a minimum of two reference genes are to be used and we determined that the most suitable seem to be PPIA and GAPD.
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Affiliation(s)
- Mirosław Andrusiewicz
- Department of Cell Biology, Health Sciences Faculty, Poznan University of Medical Sciences, Rokietnicka str. 5D, 60-806 Poznan, Poland.
| | - Bartosz Słowikowski
- Department of Cell Biology, Health Sciences Faculty, Poznan University of Medical Sciences, Rokietnicka str. 5D, 60-806 Poznan, Poland; Department of Biochemistry and Molecular Biology, Faculty of Medicine I, Poznan University of Medical Sciences, Swiecickiego str. 6, 60-781 Poznan, Poland.
| | - Izabela Skibińska
- Department of Cell Biology, Health Sciences Faculty, Poznan University of Medical Sciences, Rokietnicka str. 5D, 60-806 Poznan, Poland.
| | - Maria Wołuń-Cholewa
- Department of Cell Biology, Health Sciences Faculty, Poznan University of Medical Sciences, Rokietnicka str. 5D, 60-806 Poznan, Poland.
| | - Anna Dera-Szymanowska
- Department of Perinatology and Gynecology, Faculty of Medicine II, Poznan University of Medical Sciences, Polna Street 33, 60-535 Poznan, Poland.
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Muro S, Miyake Y, Kato H, Tsutsumi K, Yamamoto K. Serum anti-60S ribosomal protein L29 antibody as a novel prognostic marker for unresectable pancreatic cancer. Digestion 2015; 91:164-73. [PMID: 25765324 DOI: 10.1159/000371545] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/10/2014] [Accepted: 12/12/2014] [Indexed: 02/04/2023]
Abstract
BACKGROUND/AIMS Recently, we found the presence of anti-60S ribosomal protein L29 antibody (anti-RPL29) in human sera, inhibiting the proliferation of pancreatic cancer cells in vitro. We aimed to estimate the association of serum anti- RPL29 levels with clinical features in patients affected with unresectable pancreatic cancer. METHODS We retrospectively reviewed 105 patients with unresectable pancreatic cancer. Serum anti-RPL29 levels were measured by the indirect enzyme-linked immunosorbent assay. The cut-off was represented by the 95th percentile in 62 healthy volunteers. RESULTS Median survival time (MST) was 11.1 months in 49 patients showing serum anti-RPL29 level >cut-off and 7.4 months in 56 patients showing serum anti-RPL29 level ≤ cutoff. In locally advanced disease, MST was 17.9 months in 22 patients showing serum anti-RPL29 level >cut-off and 10.0 months in 19 patients showing serum anti-RPL29 level ≤ cutoff. In metastatic disease, MST was 8.7 months in 27 patients showing serum anti-RPL29 level >cut-off and 5.9 months in 37 patients showing serum anti-RPL29 level ≤ cut-off. In the multivariate Cox proportional hazard model, serum anti- RPL29 level >cut-off, abdominal or back pain, performance status, and metastatic disease were identified as independent prognostic factors. CONCLUSION Serum anti-RPL29 levels may be a novel candidate for a prognostic marker for unresectable pancreatic cancer.
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Sharan RN, Vaiphei ST, Nongrum S, Keppen J, Ksoo M. Consensus reference gene(s) for gene expression studies in human cancers: end of the tunnel visible? Cell Oncol (Dordr) 2015; 38:419-31. [PMID: 26384826 DOI: 10.1007/s13402-015-0244-6] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/07/2015] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Gene expression studies are increasingly used to provide valuable information on the diagnosis and prognosis of human cancers. Also, for in vitro and in vivo experimental cancer models gene expression studies are widely used. The complex algorithms of differential gene expression analyses require normalization of data against a reference or normalizer gene, or a set of such genes. For this purpose, mostly invariant housekeeping genes are used. Unfortunately, however, there are no consensus (housekeeping) genes that serve as reference or normalizer for different human cancers. In fact, scientists have employed a wide range of reference genes across different types of cancer for normalization of gene expression data. As a consequence, comparisons of these data and/or data harmonizations are difficult to perform and challenging. In addition, an inadequate choice for a reference gene may obscure genuine changes and/or result in erroneous gene expression data comparisons. METHODS In our effort to highlight the importance of selecting the most appropriate reference gene(s), we have screened the literature for gene expression studies published since the turn of the century on thirteen of the most prevalent human cancers worldwide. CONCLUSIONS Based on the analysis of the data at hand, we firstly recommend that in each study the suitability of candidate reference gene(s) should carefully be evaluated in order to yield reliable differential gene expression data. Secondly, we recommend that a combination of PPIA and either GAPDH, ACTB, HPRT and TBP, or appropriate combinations of two or three of these genes, should be employed in future studies, to ensure that results from different studies on different human cancers can be harmonized. This approach will ultimately increase the depth of our understanding of gene expression signatures across human cancers.
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Affiliation(s)
- R N Sharan
- Radiation and Molecular Biology Unit, Department of Biochemistry, North-Eastern Hill University (NEHU), Shillong, 793022, India.
| | - S Thangminlal Vaiphei
- Radiation and Molecular Biology Unit, Department of Biochemistry, North-Eastern Hill University (NEHU), Shillong, 793022, India
| | - Saibadaiahun Nongrum
- Radiation and Molecular Biology Unit, Department of Biochemistry, North-Eastern Hill University (NEHU), Shillong, 793022, India
| | - Joshua Keppen
- Radiation and Molecular Biology Unit, Department of Biochemistry, North-Eastern Hill University (NEHU), Shillong, 793022, India
| | - Mandahakani Ksoo
- Radiation and Molecular Biology Unit, Department of Biochemistry, North-Eastern Hill University (NEHU), Shillong, 793022, India
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Leal MF, Astur DC, Debieux P, Arliani GG, Franciozi CES, Loyola LC, Andreoli CV, Smith MC, Pochini ADC, Ejnisman B, Cohen M. Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR. PLoS One 2015; 10:e0133323. [PMID: 26192306 PMCID: PMC4507999 DOI: 10.1371/journal.pone.0133323] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2015] [Accepted: 06/25/2015] [Indexed: 11/30/2022] Open
Abstract
The anterior cruciate ligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury) and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1) injured ACL tears and controls, and (2) ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.
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Affiliation(s)
- Mariana Ferreira Leal
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038–032, São Paulo, SP, Brazil
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, 04023–001, São Paulo, SP, Brazil
- * E-mail:
| | - Diego Costa Astur
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038–032, São Paulo, SP, Brazil
| | - Pedro Debieux
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038–032, São Paulo, SP, Brazil
| | - Gustavo Gonçalves Arliani
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038–032, São Paulo, SP, Brazil
| | | | - Leonor Casilla Loyola
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038–032, São Paulo, SP, Brazil
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, 04023–001, São Paulo, SP, Brazil
| | - Carlos Vicente Andreoli
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038–032, São Paulo, SP, Brazil
| | - Marília Cardoso Smith
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, 04023–001, São Paulo, SP, Brazil
| | - Alberto de Castro Pochini
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038–032, São Paulo, SP, Brazil
| | - Benno Ejnisman
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038–032, São Paulo, SP, Brazil
| | - Moises Cohen
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038–032, São Paulo, SP, Brazil
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Reference gene selection for real-time quantitative PCR analysis on ovarian cryopreservation by vitrification in mice. J Assist Reprod Genet 2015; 32:1277-84. [PMID: 26115720 DOI: 10.1007/s10815-015-0503-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Accepted: 05/26/2015] [Indexed: 10/23/2022] Open
Abstract
PURPOSE To ensure the correct interpretation of the results of quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) from ovarian tissue cryopreserved by vitrification, it is critical to normalize expression levels to a reference gene with stable messenger RNA (mRNA) expression in the vitrified/warmed ovarian tissue. The aim of this work was to identify suitable reference genes for qRT-PCR analysis during ovarian cryopreservation by vitrification. METHODS GeNorm, NormFinder, comparative Delta-CT, and BestKeeper were used to analyze the expression and stability of the 14 reference genes GAPDH, ABL1, ACTB, CDKN1A, GPER, GUSB, HPRT1, HSP90AB1, IPO8, PPIA, RPL4, RPL30, TBP, and UPAR. RESULTS Our results indicated that ACTB and RPL4 were relatively stable reference genes in vitrified/warmed ovaries.
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Differential p16/INK4A cyclin-dependent kinase inhibitor expression correlates with chemotherapy efficacy in a cohort of 88 malignant pleural mesothelioma patients. Br J Cancer 2015; 113:69-75. [PMID: 26057448 PMCID: PMC4647524 DOI: 10.1038/bjc.2015.187] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2014] [Revised: 04/21/2015] [Accepted: 04/28/2015] [Indexed: 12/25/2022] Open
Abstract
Background: Malignant pleural mesothelioma (MPM) is a rare and essentially incurable malignancy most often linked with occupational exposure to asbestos fibres. In common with other malignancies, the development and progression of MPM is associated with extensive dysregulation of cell cycle checkpoint proteins that modulate cell proliferation, apoptosis, DNA repair and senescence. Methods: The expression of cyclin-dependent kinase inhibitor p16/INK4A was evaluated by immunohistochemistry using tumour biopsy specimens from 88 MPM cases and a semi-quantitative score for p16/INK4A expression was obtained. Post-diagnosis survival and the survival benefit of chemotherapeutic intervention was correlated with p16/INK4A expression. Results: A low, intermediate and high score for p16/INK4A expression was observed for 45 (51.1%), 28 (31.8%) and 15 (17.1%) of the MPM cases, respectively. Those cases with intermediate or high p16/INK4A tumour expression had a significantly better post-diagnosis survival than those cases whose tumours lost p16 expression (log-rank P<0.001). Those patients with sustained p16/INK4A expression who received chemotherapy also had a better survival than those treated patients whose tumours had lost p16/INK4A expression (log-rank P<0.001). Conclusions: Sustained p16/INK4A expression predicts better post-diagnosis survival in MPM and also better survival following chemotherapeutic intervention.
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Tachaudomdach C, Kantachuvesiri S, Wongpraphairot S, Worawichawong S, Tankee P, Riengrojpitak S, Kitiyakara C. High collagen I gene expression as an independent predictor of adverse renal outcomes in lupus nephritis patients with preserved renal function. Arch Pathol Lab Med 2015; 139:378-87. [PMID: 25724035 DOI: 10.5858/arpa.2013-0511-oa] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
CONTEXT The deposition of extracellular matrix is a major pathogenic mechanism leading to fibrosis and progressive decline in renal function in patients with lupus nephritis (LN). Currently, available clinicopathologic features cannot predict renal outcome consistently. OBJECTIVE To test that the expression of renal fibrogenic genes correlates with renal fibrosis at the time of biopsy and is predictive of renal outcomes. DESIGN Renal gene expression levels of transforming growth factor β-1 (TGFB1), and collagen I (COL1) were studied by real-time multiplex quantitative polymerase chain reaction in a prospective cohort of patients with LN (n = 39). Extracellular matrix index (ECMI) and collagen I/III matrix index were measured from Picro-Sirius Red-stained slides under normal and polarized light, respectively. RESULTS After follow-up (median, 43.9 months), renal failure (50% reduction in glomerular filtration rate [GFR] or dialysis) had developed in 13 subjects. The expression levels of renal fibrogenic genes were increased as compared to controls without LN. COL1 correlated with collagen I/III matrix index at baseline. Both high expression of TGFB1 or COL1 tended to predict renal failure by univariate analysis. By multivariate analysis, high ECMI and low GFR were predictive of renal failure. In patients with baseline GFR of 60 mL/min/1.73 m(2) or greater, high renal COL1 expression was an independent (hazard ratio = 4.4, P = .04) predictor of renal failure. CONCLUSIONS High renal COL1 expression is a strong predictor of adverse renal outcome in patients with LN and preserved baseline GFR. These findings support larger prospective studies to confirm the benefits of COL1 in identifying patients at high risk of progression to renal disease.
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Affiliation(s)
- Chiraporn Tachaudomdach
- From the Molecular Medicine Graduate Program, Faculty of Science, Mahidol University (Dr Tachaudomdach), the Department of Medicine, Faculty of Medicine, Ramathibodi Hospital (Drs Kantachuvesiri and Kitiyakara), the Department of Pathology, Faculty of Medicine, Ramathibodi Hospital (Dr Worawichawong), and the Department of Pathobiology, Faculty of Medicine (Dr Riengrojpitak), Mahidol University, Bangkok, Thailand; the Department of Medicine, Prince of Songkla University, Songkla, Thailand (Dr Wongpraphairot); and the Department of Medicine, Vachira Phuket Hospital, Phuket, Thailand (Dr Tankee)
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Leal MF, Belangero PS, Figueiredo EA, Cohen C, Loyola LC, Andreoli CV, Smith MC, de Castro Pochini A, Ejnisman B, Cohen M. Identification of suitable reference genes for gene expression studies in tendons from patients with rotator cuff tear. PLoS One 2015; 10:e0118821. [PMID: 25768100 PMCID: PMC4358921 DOI: 10.1371/journal.pone.0118821] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2014] [Accepted: 01/09/2015] [Indexed: 01/17/2023] Open
Abstract
Rotator cuff tear is one of the most common causes of shoulder dysfunction. Gene expression analysis may be a useful tool for understanding tendon tears and the failure of cuff healing, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluate the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1 and TBP) using samples from the rotator cuff tendons of 28 individuals with tendon tears (3 tendons regions) and 8 controls (2 tendon regions); for the tear patients, we evaluated ruptured and non-ruptured tendon samples. The stability of the candidate reference genes was determined using the NormFinder, geNorm, BestKeeper and DataAssist software packages. Overall, HPRT1 was the best single reference gene, and HPRT1+TBP composed the best pair and HPRT1+TBP+ACTB composed the best trio of reference genes from the analysis of different groups, including the simultaneous analysis of all tissue samples. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1 and COL3A1, and no obvious differences were observed when using 2, 3 or 4 reference genes for most of the analyses. However, COL3A1 expression differed between ruptured and non-ruptured (posterior superior region) tendons of patients only when normalized by HPRT1+TBP+B2M and HPRT1+TBP. On the other hand, the comparison between these two groups using the best trio of reference genes (HPRT1+TBP+ACTB) and 4 reference genes did not revealed a significant difference in COL3A1 expression. Consequently, the use of suitable reference genes for a reliable gene expression evaluation by RT-qPCR should consider the type of tendon samples investigated. HPRT1+TBP+ACTB seems to be the best combination of reference genes for the analysis of involving different tendon samples of individuals with rotator cuff tears.
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Affiliation(s)
- Mariana Ferreira Leal
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo, SP, Brazil
- * E-mail:
| | - Paulo Santoro Belangero
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | | | - Carina Cohen
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | - Leonor Casilla Loyola
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | - Carlos Vicente Andreoli
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | - Marília Cardoso Smith
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | | | - Benno Ejnisman
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | - Moises Cohen
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil
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Liu LL, Zhao H, Ma TF, Ge F, Chen CS, Zhang YP. Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection. PLoS One 2015; 10:e0117058. [PMID: 25617865 PMCID: PMC4305315 DOI: 10.1371/journal.pone.0117058] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2014] [Accepted: 12/18/2014] [Indexed: 01/08/2023] Open
Abstract
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct), and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2) expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.
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Affiliation(s)
- Lin-Lin Liu
- Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China
| | - Hui Zhao
- Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China
| | - Teng-Fei Ma
- Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China
| | - Fei Ge
- Department of Endocrine Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China
- Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China
| | - Ce-Shi Chen
- Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China
| | - Ya-Ping Zhang
- Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China
- * E-mail:
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Bian Z, Yu Y, Quan C, Guan R, Jin Y, Wu J, Xu L, Chen F, Bai J, Sun W, Fu S. RPL13A as a reference gene for normalizing mRNA transcription of ovarian cancer cells with paclitaxel and 10-hydroxycamptothecin treatments. Mol Med Rep 2014; 11:3188-94. [PMID: 25523336 DOI: 10.3892/mmr.2014.3108] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2013] [Accepted: 12/03/2014] [Indexed: 11/06/2022] Open
Abstract
Gene transcription analysis is important in cancer research, and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) has been demonstrated to be an effective method to evaluate gene transcription in cancer. RT‑qPCR requires an internal reference gene with a consistent level of mRNA transcription across various experimental conditions. However, it has been suggested that different treatments, including anticancer therapy, may influence the transcriptional stability of internal reference genes. Paclitaxel (PTX) and 10‑hydroxycamptothecin (HCPT) are widely used to treat various types of cancer, and a suitable internal reference gene is required in order to analyze the transcription profiles of the cells following treatment. In the current study, the transcriptional stability of 30 candidate reference genes was investigated in cancer cells following treatment with PTX and HCPT. The two ovarian cancer cell lines, UACC‑1598 and SKOV3, were treated with PTX and HCPT for 24 and 48 h, and the transcriptional levels of the candidate reference genes were subsequently evaluated by RT‑qPCR analysis. The transcriptional stability of the selected genes was then analyzed using qbase+ and NormFinder software. A total of 9 genes were demonstrated to exhibit high transcriptional stability and one of these genes, ribosomal protein L13a (RPL13A), was identified to exhibit high transcriptional stability in every group. The current study identified various reference genes suitable under different circumstances, while RPL13A was indicated to be the most suitable reference gene for analyzing the transcription profile of ovarian cancer cells following treatment with PTX and HCPT.
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Affiliation(s)
- Zehua Bian
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Yang Yu
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Chao Quan
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Rongwei Guan
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Yan Jin
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Jie Wu
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Lidan Xu
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Feng Chen
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Jing Bai
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Wenjing Sun
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Songbin Fu
- Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
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Dargahi D, Swayze RD, Yee L, Bergqvist PJ, Hedberg BJ, Heravi-Moussavi A, Dullaghan EM, Dercho R, An J, Babcook JS, Jones SJ. A pan-cancer analysis of alternative splicing events reveals novel tumor-associated splice variants of matriptase. Cancer Inform 2014; 13:167-77. [PMID: 25506199 PMCID: PMC4259500 DOI: 10.4137/cin.s19435] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2014] [Revised: 10/08/2014] [Accepted: 10/09/2014] [Indexed: 12/31/2022] Open
Abstract
High-throughput transcriptome sequencing allows identification of cancer-related changes that occur at the stages of transcription, pre-messenger RNA (mRNA), and splicing. In the current study, we devised a pipeline to predict novel alternative splicing (AS) variants from high-throughput transcriptome sequencing data and applied it to large sets of tumor transcriptomes from The Cancer Genome Atlas (TCGA). We identified two novel tumor-associated splice variants of matriptase, a known cancer-associated gene, in the transcriptome data from epithelial-derived tumors but not normal tissue. Most notably, these variants were found in 69% of lung squamous cell carcinoma (LUSC) samples studied. We confirmed the expression of matriptase AS transcripts using quantitative reverse transcription PCR (qRT-PCR) in an orthogonal panel of tumor tissues and cell lines. Furthermore, flow cytometric analysis confirmed surface expression of matriptase splice variants in chinese hamster ovary (CHO) cells transiently transfected with cDNA encoding the novel transcripts. Our findings further implicate matriptase in contributing to oncogenic processes and suggest potential novel therapeutic uses for matriptase splice variants.
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Affiliation(s)
- Daryanaz Dargahi
- BC Cancer Agency, Genome Sciences Center, Vancouver, British Columbia, Canada. ; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada
| | - Richard D Swayze
- Center for Drug Research and Development (CDRD), Vancouver, British Columbia, Canada
| | - Leanna Yee
- Center for Drug Research and Development (CDRD), Vancouver, British Columbia, Canada
| | - Peter J Bergqvist
- Center for Drug Research and Development (CDRD), Vancouver, British Columbia, Canada
| | - Bradley J Hedberg
- Center for Drug Research and Development (CDRD), Vancouver, British Columbia, Canada
| | | | - Edie M Dullaghan
- Center for Drug Research and Development (CDRD), Vancouver, British Columbia, Canada
| | - Ryan Dercho
- Center for Drug Research and Development (CDRD), Vancouver, British Columbia, Canada
| | - Jianghong An
- BC Cancer Agency, Genome Sciences Center, Vancouver, British Columbia, Canada
| | - John S Babcook
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada. ; Center for Drug Research and Development (CDRD), Vancouver, British Columbia, Canada
| | - Steven Jm Jones
- BC Cancer Agency, Genome Sciences Center, Vancouver, British Columbia, Canada. ; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada
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Wierzbicki PM, Klacz J, Rybarczyk A, Slebioda T, Stanislawowski M, Wronska A, Kowalczyk A, Matuszewski M, Kmiec Z. Identification of a suitable qPCR reference gene in metastatic clear cell renal cell carcinoma. Tumour Biol 2014; 35:12473-87. [PMID: 25225161 PMCID: PMC4275580 DOI: 10.1007/s13277-014-2566-9] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2014] [Accepted: 08/27/2014] [Indexed: 11/20/2022] Open
Abstract
There is no data on reference gene (RG) selection in metastatic clear-cell renal cell carcinoma (mccRCC) for quantitative PCR (qPCR) data normalization. We aimed at selecting the most stable RG for further determination of new prognostic markers. Thirty-five nonmetastatic and 35 mccRCC patients undergoing radical nephrectomy were included. Paired primary tumor (T, n = 70) and normal (C, n = 70) kidney fragments were collected; from 12 out of 35 mccRCC cases, we also collected metastasized regional lymph nodes and adrenal gland tissues (M, n = 12). After RNA extraction, reverse transcription and qPCR were performed. Samples were divided into four analyzed groups. Fifteen candidate RGs were tested by RefFinder tool and manual statistics. To present the importance of RG selection, TP53 gene expression levels in samples were normalized with the use of RG data. RPL13 gene was the most stable RG in analysis of 35 primary tumor nonmetastatic versus 35 mccRCC samples and matched metastasized T/C/M samples (n = 12, each group). GUSB was the most suitable RG in total 152 samples and in paired T and C (n = 140) kidney samples. Expression of GUSB, RPL13, and the RPL13 + RPLP0 pair were independent of clinical/sample variables. Normalization of TP53 expression levels showed variability of GAPDH and ACTB assays. GUSB or RPL13 assays should be used in mccRCC for qPCR data normalization whereas GAPDH and ACTB assays should be avoided. Prior RG studies should precede each qPCR gene expression study since RG selection is associated with the origin and proportion of specimens.
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Affiliation(s)
- Piotr M Wierzbicki
- Department of Histology, Faculty of Medicine, Medical University of Gdansk, ul. Dębinki 1, PL 80-211, Gdańsk, Poland,
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Yu S, Yang Q, Yang JH, Du Z, Zhang G. Identification of suitable reference genes for investigating gene expression in human gallbladder carcinoma using reverse transcription quantitative polymerase chain reaction. Mol Med Rep 2014; 11:2967-74. [PMID: 25434674 DOI: 10.3892/mmr.2014.3008] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2014] [Accepted: 11/05/2014] [Indexed: 11/06/2022] Open
Abstract
Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT‑qPCR data. The key aim of this method is to identify an applicable internal reference gene, however, there are currently no suitable reference genes for gene analysis in gallbladder carcinoma. In the present study, screening was performed using 12 common reference genes, which were selected in order to provide an experimental basis for the investigation of gene expression in gallbladder carcinoma. A total of 16 tissue samples of gallbladder carcinoma and their matched normal gallbladder tissues were used. The gene expression stability and applicability of the 12 reference gene candidates were determined using the geNorm, NormFinder and BestKeeper software programs. Following comparison of the results of the three software programs, HPRT1 was identified as the most stably expressed reference gene. In the normal gallbladder group, the relative stably expressed reference gene was PPIA and in the entire sample group, the relatively stably expressed reference gene was PPIA. The present study also demonstrated that the combination of the three reference genes was the most appropriate. The recommended combinations were PPIA + PUM1 + ACTB for the total sample group, GAPDH + PBGD + ALAS1 for the gallbladder carcinoma group and PPIA + PUM1 + TBP for the paired normal gallbladder group.
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Affiliation(s)
- Shan Yu
- Department of Neurology, China‑Japan Union Hospital, Jilin University, Changchun, Jilin 130033, P.R. China
| | - Qiwei Yang
- Central Laboratory, Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China
| | - Jing Hui Yang
- Department of Hepatopancreatobiliary Surgery, China‑Japan Union Hospital, Jilin University, Changchun, Jilin 130033, P.R. China
| | - Zhenwu Du
- Central Laboratory, Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China
| | - Guizhen Zhang
- Central Laboratory, Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China
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Leal MF, Belangero PS, Cohen C, Figueiredo EA, Loyola LC, Pochini AC, Smith MC, Andreoli CV, Belangero SI, Ejnisman B, Cohen M. Identification of suitable reference genes for gene expression studies of shoulder instability. PLoS One 2014; 9:e105002. [PMID: 25122470 PMCID: PMC4133370 DOI: 10.1371/journal.pone.0105002] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2014] [Accepted: 07/15/2014] [Indexed: 11/29/2022] Open
Abstract
Shoulder instability is a common shoulder injury, and patients present with plastic deformation of the glenohumeral capsule. Gene expression analysis may be a useful tool for increasing the general understanding of capsule deformation, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. Although RT-qPCR is highly sensitive and specific, it requires the use of suitable reference genes for data normalization to guarantee meaningful and reproducible results. In the present study, we evaluated the suitability of a set of reference genes using samples from the glenohumeral capsules of individuals with and without shoulder instability. We analyzed the expression of six commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, TBP and TFRC) in the antero-inferior, antero-superior and posterior portions of the glenohumeral capsules of cases and controls. The stability of the candidate reference gene expression was determined using four software packages: NormFinder, geNorm, BestKeeper and DataAssist. Overall, HPRT1 was the best single reference gene, and HPRT1 and B2M composed the best pair of reference genes from different analysis groups, including simultaneous analysis of all tissue samples. GenEx software was used to identify the optimal number of reference genes to be used for normalization and demonstrated that the accumulated standard deviation resulting from the use of 2 reference genes was similar to that resulting from the use of 3 or more reference genes. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1. Although the use of different reference gene combinations yielded variable normalized quantities, the relative quantities within sample groups were similar and confirmed that no obvious differences were observed when using 2, 3 or 4 reference genes. Consequently, the use of 2 stable reference genes for normalization, especially HPRT1 and B2M, is a reliable method for evaluating gene expression by RT-qPCR.
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Affiliation(s)
- Mariana Ferreira Leal
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Ferderal de São Paulo, São Paulo, São Paulo, Brazil
- * E-mail:
| | - Paulo Santoro Belangero
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
| | - Carina Cohen
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
| | - Eduardo Antônio Figueiredo
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
| | - Leonor Casilla Loyola
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Ferderal de São Paulo, São Paulo, São Paulo, Brazil
| | - Alberto Castro Pochini
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
| | - Marília Cardoso Smith
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Ferderal de São Paulo, São Paulo, São Paulo, Brazil
| | - Carlos Vicente Andreoli
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
| | - Sintia Iole Belangero
- Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Ferderal de São Paulo, São Paulo, São Paulo, Brazil
- Laboratório Interdisciplinar de Neurociência Clínica, Departamento de Psiquiatria, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
| | - Benno Ejnisman
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
| | - Moises Cohen
- Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
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Swierzko AS, Szala A, Sawicki S, Szemraj J, Sniadecki M, Sokolowska A, Kaluzynski A, Wydra D, Cedzynski M. Mannose-Binding Lectin (MBL) and MBL-associated serine protease-2 (MASP-2) in women with malignant and benign ovarian tumours. Cancer Immunol Immunother 2014; 63:1129-40. [PMID: 25038892 PMCID: PMC4209098 DOI: 10.1007/s00262-014-1579-y] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2013] [Accepted: 07/02/2014] [Indexed: 11/28/2022]
Abstract
Mannose-Binding Lectin (MBL) is a serum pattern recognition molecule, able to activate complement in association with MASP proteases. Serum levels of MBL and MASP-2, activities of MBL-MASP complexes, single nucleotide polymorphisms of the MBL2 and MASP2 genes and/or their specific mRNA expression in ovarian sections were investigated in 128 patients suffering from primary ovarian cancer (OC) and compared with 197 controls (C), encompassing both patients with benign ovarian tumours (n = 123) and others with no ovarian pathology (n = 74). MBL deficiency-associated genotypes were more common among OC patients than among controls. The O/O group of genotypes was associated with ovarian cancer (OR 3.5, p = 0.02). In A/A homozygotes, MBL concentrations and activities were elevated in the OC group and correlated with C-reactive protein. Moreover, high MBL serum levels were associated with more advanced disease stage. No differences in distribution of the MASP2 +359 A>G (D120G) SNP or MASP-2 serum levels were found between cancer patients and their controls. However, the highest frequency of the A/G (MASP2) and LXA/O or O/O (MBL2) genotypes was found among OC patients with tumours of G1-2 grade (well/moderately differentiated). Furthermore, MBL deficiency-associated genotypes predicted prolonged survival. None of the parameters investigated correlated with CA125 antigen or patients' age. The local expression of MBL2 and MASP2 genes was higher in women with ovarian cancer compared with controls. It is concluded that the expression of MBL and MASP-2 is altered in ovarian cancer, possibly indicating involvement of the lectin pathway of complement activation in the disease.
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Affiliation(s)
- Anna St Swierzko
- Laboratory of Immunobiology of Infections, Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, 93-232, Lodz, Poland
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Kim HJ, Na JI, Min BW, Na JY, Lee KH, Lee JH, Lee YJ, Kim HS, Park JT. Evaluation of Protein Expression in Housekeeping Genes across Multiple Tissues in Rats. KOREAN JOURNAL OF PATHOLOGY 2014; 48:193-200. [PMID: 25013417 PMCID: PMC4087132 DOI: 10.4132/koreanjpathol.2014.48.3.193] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Revised: 03/28/2014] [Accepted: 04/21/2014] [Indexed: 11/18/2022]
Abstract
Background Housekeeping genes, which show constant protein expression patterns between different tissue types, are very important in molecular biological studies as an internal control for protein research. Methods The protein expression profiles of seven housekeeping genes (HPRT1, PPIA, GYS1, TBP, YWHAZ, GAPDH and ACTB) in various rat tissues (cerebrum, cerebellum, cardiac ventricle and atrium, psoas muscle, femoral muscle, liver, spleen, kidney, and aorta) were analyzed by Western blot and compared by coefficient of variation (CV). Results HPRT1 was stably expressed (CV≤10%) in six tissues (cerebrum, cerebellum, ventricle, femoral muscle, spleen, and kidney), PPIA was stably expressed in five tissues (cerebrum, cerebellum, ventricle, spleen and kidney), YWHAZ was stably expressed in three tissues (cerebrum, cerebellum, and kidney), and GAPDH was stably expressed in four tissues (cerebrum, ventricle, psoas muscle, and kidney). In comparison, GYS1, TBP, and ACTB were found to have CV values over 10% in all tissues. Of the seven genes examined, four (HPRT1, PPIA, YWHAZ, and GAPDH) were found to be stably expressed across multiple organs, with low CV values (≤10%). Conclusions These results will provide fundamental information regarding internal controls for protein expression studies and can be used for analysis of postmortem protein degradation patterns in forensic medicine.
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Affiliation(s)
- Hye Jeong Kim
- Department of Forensic Medicine, Chonnam National University Medical School, Gwangju, Korea
| | - Jong In Na
- Department of Forensic Medicine, Chonnam National University Medical School, Gwangju, Korea
| | - Byung Woo Min
- Department of Forensic Medicine, Chonnam National University Medical School, Gwangju, Korea
| | - Joo Young Na
- Department of Forensic Medicine, Chonnam National University Medical School, Gwangju, Korea
| | - Kyung Hwa Lee
- Department of Pathology, Chonnam National University Medical School, Gwangju, Korea
| | - Jae Hyuk Lee
- Department of Pathology, Chonnam National University Medical School, Gwangju, Korea
| | - Young Jik Lee
- Department of Forensic Medicine, Chonnam National University Medical School, Gwangju, Korea
| | - Hyung Seok Kim
- Department of Forensic Medicine, Chonnam National University Medical School, Gwangju, Korea
| | - Jong Tae Park
- Department of Forensic Medicine, Chonnam National University Medical School, Gwangju, Korea
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Cai J, Li T, Huang B, Cheng H, Ding H, Dong W, Xiao M, Liu L, Wang Z. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples. PLoS One 2014; 9:e95974. [PMID: 24776823 PMCID: PMC4002476 DOI: 10.1371/journal.pone.0095974] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2014] [Accepted: 04/01/2014] [Indexed: 11/19/2022] Open
Abstract
Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.
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Affiliation(s)
- Jing Cai
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Tao Li
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Bangxing Huang
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Henghui Cheng
- Department of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Hui Ding
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Weihong Dong
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Man Xiao
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Ling Liu
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Zehua Wang
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- * E-mail:
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47
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ZHANG JUAN, TANG HONGJU, ZHANG YUQING, DENG RUYUAN, SHAO LI, LIU YUN, LI FENGYING, WANG XIAO, ZHOU LIBIN. Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation. Int J Mol Med 2014; 33:1209-18. [DOI: 10.3892/ijmm.2014.1695] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2013] [Accepted: 02/27/2014] [Indexed: 11/06/2022] Open
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48
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Braga LDC, Silva LM, Ramos APÁDS, Piedade JB, Vidigal PVT, Traiman P, da Silva Filho AL. Single CpG island methylation is not sufficient to maintain the silenced expression of CASPASE-8 apoptosis-related gene among women with epithelial ovarian cancer. Biomed Pharmacother 2013; 68:87-91. [PMID: 24412083 DOI: 10.1016/j.biopha.2013.12.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2013] [Accepted: 12/10/2013] [Indexed: 12/15/2022] Open
Abstract
Despite impressive research efforts, the biology of epithelial ovarian cancer (EOC) remains poorly understood and alterations in the expression of CASPASE-8 contribute to a worse tumor prognosis. This study assesses the methylation of the CpG island within the CASPASE-8 promoter and CASPASE-8 gene expression both in cystadenoma tumors and in primary and metastatic EOC. DNA and RNA were obtained from women with normal ovarian tissues (n=18), ovarian serous cystadenoma tumors (n=11) and EOC (n=16) using Trizol(®). The methylation frequency of the CpG island in the CASPASE-8 promoter was assessed using the methylation-specific PCR assay after DNA bisulfite conversion. Quantitative PCR was performed to quantify the relative levels of CASPASE-8 in each sample. The differences between samples with each group were evaluated using the Mann-Whitney U and Kruskal-Wallis tests as indicated. Hemimethylation of the CASPASE-8 promoter was found in 11.8% of the normal ovary samples, 20% of the cystadenoma tumors and 20% of the metastatic EOC, while methylation of the CASPASE-8 promoter was absent in the EOC primary tissues (P=0.047). An increased CASPASE-8 expression level was observed in all tumor groups. Significant differences were observed in the CASPASE-8 expression levels when compared with all ovarian tumor groups (P=0.0278). Promoter DNA methylation did not associate with expression levels of CASPASE-8, suggesting the presence of other mechanisms in relation to gene expression control in EOC; thus providing a better understanding of this complex disease.
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Affiliation(s)
- Letícia da Conceição Braga
- Serviço de Biologia Celular, Diretoria de Pesquisa e Desenvolvimento da Fundação Ezequiel Dias, Belo Horizonte, M.G., Brazil; Departamento de Ginecologia e Obstetrícia da Faculdade de Medicina da Universidade Estadual de São Paulo "Júlio de Mesquita Filho", Botucatu, S.P., Brazil
| | - Luciana Maria Silva
- Serviço de Biologia Celular, Diretoria de Pesquisa e Desenvolvimento da Fundação Ezequiel Dias, Belo Horizonte, M.G., Brazil
| | | | - Josiane Barbosa Piedade
- Serviço de Biologia Celular, Diretoria de Pesquisa e Desenvolvimento da Fundação Ezequiel Dias, Belo Horizonte, M.G., Brazil
| | - Paula Vieira Teixeira Vidigal
- Departamento de Patologia e Medicina Legal da Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, M.G., Brazil
| | - Paulo Traiman
- Departamento de Ginecologia e Obstetrícia da Faculdade de Medicina da Universidade Estadual de São Paulo "Júlio de Mesquita Filho", Botucatu, S.P., Brazil
| | - Agnaldo Lopes da Silva Filho
- Departamento de Ginecologia e Obstetrícia da Faculdade de Medicina da Universidade Federal de Minas Gerais, Avenida Professor Alfredo Balena, 190, Santa Efigênia, Belo Horizonte, M.G., Brazil; Departamento de Ginecologia e Obstetrícia da Faculdade de Medicina da Universidade Estadual de São Paulo "Júlio de Mesquita Filho", Botucatu, S.P., Brazil.
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Zheng G, Wang H, Zhang X, Yang Y, Wang L, Du L, Li W, Li J, Qu A, Liu Y, Wang C. Identification and validation of reference genes for qPCR detection of serum microRNAs in colorectal adenocarcinoma patients. PLoS One 2013; 8:e83025. [PMID: 24349425 PMCID: PMC3859607 DOI: 10.1371/journal.pone.0083025] [Citation(s) in RCA: 87] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2013] [Accepted: 11/06/2013] [Indexed: 12/14/2022] Open
Abstract
Serum microRNAs (miRNAs) have become a highlighted research hotspot, especially for their great potential as a novel promising non-invasive biomarker in cancer diagnosis. The most frequently used approach for serum miRNAs detection is quantitative real time polymerase chain reaction (qPCR). In order to obtain reliable qPCR data of miRNAs expression, the use of reference genes as endogenous control is undoubtly necessary. However, no systematic evaluation and validation of reference genes for normalizing qPCR analysis of serum miRNAs has been reported in colorectal adenocarcinoma. We firstly profiled pooled serum of colorectal adenocarcinoma, colorectal adenoma and healthy controls and selected a list of 13 miRNAs as candidate reference genes. U6 snRNA (U6) and above-mentioned 13 miRNAs were included in further confirmation by qPCR. As a result, 5 miRNAs (miR-151a-3p, miR-4446-3p, miR-221-3p, miR-93-5p and miR-3184-3p) were not detected in all samples and 2 miRNAs (miR-197-3p and miR-26a-5p) were relatively low with median Cq more than 35, and were excluded from further stability analysis. Then variable stability of other 6 miRNAs (miR-103b, miR-484, miR-16-5p, miR-3615, miR-18a-3p and miR-191-5p) and U6 were evaluated using two algorithms: geNorm and NormFinder which both identified miR-191-5p as the most stably expressed reference gene and selected miR-191-5p and U6 as the most stable pair of reference genes. After validating in an independent large cohorts and selecting miR-92a-3p as target miRNA to evaluate the effect of reference gene, we propose that combination of miR-191-5p and U6 could be used as reference genes for serum microRNAs qPCR data in colorectal adenocarcinoma, colorectal adenoma and healthy controls.
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Affiliation(s)
- Guixi Zheng
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Haiyan Wang
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Xin Zhang
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Yongmei Yang
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Lili Wang
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Lutao Du
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Wei Li
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Juan Li
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Ailin Qu
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Yimin Liu
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
| | - Chuanxin Wang
- Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
- * E-mail:
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50
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Wisnieski F, Calcagno DQ, Leal MF, Santos LCD, Gigek CDO, Chen ES, Pontes TB, Assumpção PP, Assumpção MBD, Demachki S, Burbano RR, Smith MDAC. Reference genes for quantitative RT-PCR data in gastric tissues and cell lines. World J Gastroenterol 2013; 19:7121-7128. [PMID: 24222956 PMCID: PMC3819548 DOI: 10.3748/wjg.v19.i41.7121] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/05/2013] [Revised: 08/06/2013] [Accepted: 08/20/2013] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate the suitability of reference genes in gastric tissue samples and cell lines.
METHODS: The suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent non-neoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The ranking of the best single and combination of reference genes was determined by NormFinder, geNorm™, BestKeeper, and DataAssist™. In addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization.
RESULTS: ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. The level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44).
CONCLUSION: GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested.
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