The development of new drugs is multidisciplinary and systematic work. High-throughput techniques based on “-omics” have driven the discovery of biomarkers in diseases and therapeutic targets of drugs. A transcriptome is the complete set of all RNAs transcribed by certain tissues or cells at a specific stage of development or physiological condition. Transcriptome research can demonstrate gene functions and structures from the whole level and reveal the molecular mechanism of specific biological processes in diseases. Currently, gene expression microarray and high-throughput RNA-sequencing have been widely used in biological, medical, clinical, and drug research. The former has been applied in drug screening and biomarker detection of drugs due to its high throughput, fast detection speed, simple analysis, and relatively low price. With the further development of detection technology and the improvement of analytical methods, the detection flux of RNA-seq is much higher but the price is lower, hence it has powerful advantages in detecting biomarkers and drug discovery. Compared with the traditional RNA-seq, scRNA-seq has higher accuracy and efficiency, especially the single-cell level of gene expression pattern analysis can provide more information for drug and biomarker discovery. Therefore, (sc)RNA-seq has broader application prospects, especially in the field of drug discovery. In this overview, we will review the application of these technologies in drug, especially in natural drug and biomarker discovery and development. Emerging applications of scRNA-seq and the third generation RNA-sequencing tools are also discussed.

• RNA-seq
• biomarker discovery
• gene expression microarray
• natural drug discovery
• transcriptome

• Blastomere
• Cell lineage
• Gene networks
• Human embryos

• Blastocyst
• Blastomeres/metabolism
• Cell Differentiation/genetics
• Cell Lineage/genetics
• Embryo, Mammalian/cytology
• Embryonic Development/genetics
• Epigenesis, Genetic
• Gene Expression Regulation, Developmental/genetics
• Gene Regulatory Networks/genetics
• Genome, Human/genetics
• Humans
• Systems Biology/methods

• G0801057 Medical Research Council
• MR/L004992/1 Medical Research Council
• FP7 Ideas: European Research Council

• DNA, Complementary/genetics
• RNA/genetics
• Sequence Analysis, RNA/methods
• Single-Cell Analysis/methods
• Transcriptome

• single-cell
• heterogeneity
• transcriptomics
• rna-seq
• rna sequencing
• microarrays
• rt-qpcr

• Gene Expression Profiling/methods
• Reverse Transcriptase Polymerase Chain Reaction/methods
• Sequence Analysis, RNA/methods
• Single-Cell Analysis/methods

• characterization
• flow cytometry
• glaucoma
• mesenchymal stem cells
• regenerative medicine
• trabecular meshwork

• Noise
• RNA-seq
• Single cell
• Variability

• Animals
• Gene Expression Profiling/methods
• Humans
• Single-Cell Analysis/methods

• BB/J004235/1 Biotechnology and Biological Sciences Research Council

Single cell sequencing (SCS) has become a new approach to study biological heterogeneity. The advancement in technologies for single cell isolation, amplification of genome/transcriptome and next-generation sequencing enables SCS to reveal the inherent properties of a single cell from the large scale of the genome, transcriptome or epigenome at high resolution. Recently, SCS has been widely applied in various clinical and research fields, such as cancer biology and oncology, immunology, microbiology, neurobiology and prenatal diagnosis. In this review, we will discuss the development of SCS methods and focus on the latest clinical and research applications of SCS.

• alpha cell marker
• pancreas development
• single cell RNA-Seq

• axons
• lamprey
• mRNA
• regeneration
• ribosomes
• spinal cord

• Animals
• Cells/classification
• Cells/metabolism
• Gene Expression Profiling
• Guidelines as Topic
• Humans
• Sequence Analysis, DNA
• Sequence Analysis, RNA
• Single-Cell Analysis

It has been believed that mammalian adult cardiomyocytes (ACMs) are terminally-differentiated and are unable to proliferate. Recently, using a bi-transgenic ACM fate mapping mouse model and an in vitro culture system, we demonstrated that adult mouse cardiomyocytes were able to dedifferentiate into cardiac progenitor-like cells (CPCs). However, little is known about the molecular basis of their intrinsic cellular plasticity. Here we integrate single-cell transcriptome and whole-genome DNA methylation analyses to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse ACMs. Compared to parental cardiomyocytes, dedifferentiated mouse cardiomyocyte-derived CPCs (mCPCs) display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlated well with the methylome, our transcriptomic data showed that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implantation of mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. Our study demonstrates that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration.

• Analysis
• RT
• amplification qPCR
• harvesting
• single-cell

• Cell Separation/instrumentation
• Cell Separation/methods
• Electrophoresis, Capillary/instrumentation
• Electrophoresis, Capillary/methods
• Escherichia coli/chemistry
• Flow Cytometry/instrumentation
• Flow Cytometry/methods
• Gene Expression Profiling/instrumentation
• Gene Expression Profiling/methods
• Gene Expression Regulation
• Humans
• Laser Capture Microdissection/instrumentation
• Laser Capture Microdissection/methods
• Microfluidics/instrumentation
• Microfluidics/methods
• Phenotype
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• Real-Time Polymerase Chain Reaction/instrumentation
• Real-Time Polymerase Chain Reaction/methods
• Reverse Transcription
• Single-Cell Analysis/instrumentation
• Single-Cell Analysis/methods
• Thermus/chemistry

• Genomics
• Lineage specification
• Mouse embryo
• Quantitative high-resolution imaging
• Single-cell analysis
• Systems biology

• Cortex
• Ephrin A5
• Neurogenesis
• Thalamus

• single cells
• transcriptome
• rna-seq
• cancer stem cells
• embryonic stem cells

• Embryo, Mammalian/metabolism
• Embryonic Development/genetics
• Gene Expression Profiling/methods
• Humans
• Polymerase Chain Reaction
• Preimplantation Diagnosis/methods
• Single-Cell Analysis/methods
• Stem Cells/metabolism
• Transcriptome

• P01 GM099130 NIGMS NIH HHS
• 1P01GM099130-01 NIGMS NIH HHS

DNA microarrays are widely used as end point detectors for gene expression analysis. Several methods have been developed for target labelling to enable quantification but without taking target length into consideration. Here we highlight the importance of choosing the optimum target length that would ensure specificity without compromising sensitivity of the assay. For this, eight plasmids that are identical to each other except for a closely related 23 bp unique reporter (UR) sequence were used to examine the hybridization efficiency for these URs. Targets of various lengths were generated and labelled as follows: full length and 330 bases transcripts using a dendrimer labelling method, 120 bp amplicons by the modified PCR end labelling method and synthetic labelled targets of 33 bases. This report also shows the advantages of using the modified PCR method over other labelling methods in generating labelled amplicons of the desired lengths to maximize hybridization efficiency.

• Dendrimer.
• Hybridization specificity
• Oligonucleotide microarrays
• PCR
• Secondary structure
• Sensitivity

Advances in whole-genome and whole-transcriptome amplification have permitted the sequencing of the minute amounts of DNA and RNA present in a single cell, offering a window into the extent and nature of genomic and transcriptomic heterogeneity which occurs in both normal development and disease. Single-cell approaches stand poised to revolutionise our capacity to understand the scale of genomic, epigenomic, and transcriptomic diversity that occurs during the lifetime of an individual organism. Here, we review the major technological and biological breakthroughs achieved, describe the remaining challenges to overcome, and provide a glimpse into the promise of recent and future developments.

• DNA, Complementary/genetics
• DNA-Directed DNA Polymerase/genetics
• Gene Expression Profiling/methods
• Genome, Plant
• Nucleic Acid Amplification Techniques/methods
• RNA/chemistry
• RNA/genetics
• Solanum tuberosum/genetics

• Adult
• Female
• Gene Expression Profiling
• Gene Library
• Humans
• RNA, Bacterial/analysis
• RNA, Bacterial/genetics
• Sexual Behavior
• Vaginosis, Bacterial/diagnosis

Early development in humans is characterised by low and variable embryonic viability, reflected in low fecundity and high rates of miscarriage, relative to other mammals. Data from assisted reproduction programmes provides additional evidence that this is largely mediated at the level of embryonic competence and is highly heterogeneous among embryos. Understanding the basis of this heterogeneity has important implications in a number of areas including: the regulation of early human development, disorders of pregnancy, assisted reproduction programmes, the long term health of children which may be programmed in early development, and the molecular basis of pluripotency in human stem cell populations. We have therefore investigated global gene expression profiles using polyAPCR amplification and microarray technology applied to individual human oocytes and 4-cell and blastocyst stage embryos. In order to explore the basis of any variability in detail, each developmental stage is replicated in triplicate. Our data show that although transcript profiles are highly stage-specific, within each stage they are relatively variable. We describe expression of a number of gene families and pathways including apoptosis, cell cycle and amino acid metabolism, which are variably expressed and may be reflective of embryonic developmental competence. Overall, our data suggest that heterogeneity in human embryo developmental competence is reflected in global transcript profiles, and that the vast majority of existing human embryo gene expression data based on pooled oocytes and embryos need to be reinterpreted.

• Amino Acids/metabolism
• Apoptosis
• Carbohydrate Metabolism
• Cell Cycle
• Embryo, Mammalian/cytology
• Embryo, Mammalian/metabolism
• Extracellular Matrix/metabolism
• Focal Adhesions
• Gene Expression Profiling
• Humans
• Oocytes/metabolism
• Polymerase Chain Reaction/methods
• Progesterone/physiology
• Purines/metabolism
• Pyrimidines/metabolism
• Signal Transduction
• Transforming Growth Factor beta/metabolism
• Tumor Suppressor Protein p53/metabolism

• G0300484 Medical Research Council
• G0700092 Medical Research Council
• G0801057 Medical Research Council
• C19686/A6233 Cancer Research UK

In the last decade, genome-wide transcriptome analyses have been routinely used to monitor tissue-, disease- and cell type-specific gene expression, but it has been technically challenging to generate expression profiles from single cells. Here we describe a novel and robust mRNA-Seq protocol (Smart-Seq) that is applicable down to single cell levels. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which significantly enhances detailed analyses of alternative transcript isoforms and identification of SNPs. We have determined the sensitivity and quantitative accuracy of Smart-Seq for single-cell transcriptomics by evaluating it on total RNA dilution series. Applying Smart-Seq to circulating tumor cells from melanomas, we identified distinct gene expression patterns, including new candidate biomarkers for melanoma circulating tumor cells. Importantly, our protocol can easily be utilized for addressing fundamental biological problems requiring genome-wide transcriptome profiling in rare cells.

Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them.

Understanding brain function involves improved knowledge about how the genome specifies such a large diversity of neuronal types. Transcriptome analysis of single neurons has been previously described using gene expression microarrays. Using high-throughput transcriptome sequencing (RNA-Seq), we have developed a method to perform single-neuron RNA-Seq. Following electrophysiology recording from an individual neuron, total RNA was extracted by aspirating the cellular contents into a fine glass electrode tip. The mRNAs were reverse transcribed and amplified to construct a single-neuron cDNA library, and subsequently subjected to high-throughput sequencing. This approach was applied to both individual neurons cultured from embryonic mouse hippocampus, as well as neocortical neurons from live brain slices. We found that the average pairwise Spearman’s rank correlation coefficient of gene expression level expressed as RPKM (reads per kilobase of transcript per million mapped reads) was 0.51 between five cultured neuronal cells, whereas the same measure between three cortical layer 5 neurons in situ was 0.25. The data suggest that there may be greater heterogeneity of the cortical neurons, as compared to neurons in vitro. The results demonstrate the technical feasibility and reproducibility of RNA-Seq in capturing a part of the transcriptome landscape of single neurons, and confirmed that morphologically identical neurons, even from the same region, have distinct gene expression patterns.

• RNA-Seq
• cell culture
• electrophysiology
• gene expression
• neuron
• transcriptome

• Animals
• Brain/cytology
• Cell Separation
• Drosophila melanogaster/cytology
• Drosophila melanogaster/genetics
• Gene Expression Profiling/methods
• Larva/cytology
• Larva/genetics
• Needles
• Neural Stem Cells/cytology
• Neural Stem Cells/metabolism
• Polymerase Chain Reaction
• Reverse Transcription

• 092096 Wellcome Trust

• Animals
• Cell Differentiation/genetics
• Gene Expression Profiling
• Genotype
• Humans
• Mice
• Retinoblastoma/genetics
• Retinoblastoma/pathology

• R01 EY018599 NEI NIH HHS
• Howard Hughes Medical Institute
• R01 CA168875 NCI NIH HHS
• CA114102 NCI NIH HHS
• EY018599 NEI NIH HHS
• EY014867 NEI NIH HHS
• CA21765 NCI NIH HHS
• R01 EY014867 NEI NIH HHS
• P30 CA021765 NCI NIH HHS
• R01 CA114102 NCI NIH HHS

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy.

• Animals
• DNA, Complementary/analysis
• DNA, Complementary/metabolism
• DNA-Directed RNA Polymerases/metabolism
• Electroporation/methods
• Escherichia coli/genetics
• Gene Expression
• Gene Expression Profiling
• Gene Library
• Genetic Association Studies
• Microarray Analysis
• Nucleic Acid Amplification Techniques/methods
• RNA, Messenger/analysis
• RNA, Messenger/chemistry
• Reverse Transcriptase Polymerase Chain Reaction
• Ribonucleases/metabolism
• Transcription, Genetic
• Viral Proteins/metabolism