Cross-species hybridization of woodchuck hepatitis virus-induced hepatocellular carcinoma using human oligonucleotide microarrays

Table 1 RT-PCR primer and probe sequences for select genes

Target gene	Forward primer sequence	Reverse primer sequence	Reporter probe sequence
Testis enhanced gene transcript (BAX inhibitor 1) (TEGT)	GCCACCTGTAATGTGTGCAAAG	ACTTTCCCAGTTTGACTTCCTCTTC	CTCCACCCCTCATGTCC
Acetyl-CoA synthetase 2 (thiokinase) (ACAS2)	TGCTGAGGACCCACTCTTCAT	AGCATGTAACCGCCAACTGT	CCACAGGCAAACCCA
Alpha-fetoprotein (AFP)	AGGCTGTCATTGCAGATTTCTCT	GGACCCTCTTCTGCAAAGCA	CTGGCAGCATGTCTCC
C-myc (CMYC)	CAGAAGGTCAGAATCGGTGTCA	AGGACCAGTGGGCTGTGA	CCACAGCAAACCTC
Hexokinase (glucokinase regulatory protein) (GCKR)	ATGCTGCAGCGGTTCTCT	GGATCGCTTGGAGGAGACTCT	AAGGCCCGATGCATTG
Isocitrate dehydrogenase 3 (NAD+) beta (IDH3B)	TCTCAGCGGATTGCAAAGTTTG	CTTGTGGACAGCTGTGACCTT	CCCGCCCCTTCTTG
Fatty-acid-Coenzyme A ligase, very long-chain 1 (FACVL1)	GCGGGATGACACAGCAAAA	TCTTTCTGGAAATGTGAGTTATCCTTCTG	TCTGAAAATCTGAATATCCC
Spermidine/spermine N1-acetyltransferase (SSAT)	TTTCATGCAACACTTGGTCTCTCT	CACTGGACTCCGGAAGGTAAC	CCCTCACCCAATCCAG
Carnitine palmitoyltransferase II (CPT2)	TGCCTATTCCCAAACTTGAAGACA	TCTTCCTGAACTGGCCATCATTC	TCAATGCACAGAAACCT
N-myc 2 (NMYC2)	GGACGCGCTGAGTGGAT	GCTCTCGTTCGGCTCCAA	TTCCCAGGAGACCCC

Table 2 Quality control data for cross-species hybridization

Samples	RNA integrity	Average	Noise	Percentage of	Scaling
	number	background		genes called	factor
	(max = 10.0)			present	(scaled to 15)
W904Nb	7.5	33.750	1.00	9.0	2.959
W361N	7.8	34.170	0.97	9.5	2.970
W380N	9.7	32.089	0.93	9.9	2.955
W904T	9.8	36.880	1.07	10.3	2.206
W361T	9.2	35.720	1.05	10.5	2.355
W380T	9.9	31.610	0.94	10.8	2.463

Table 3 Significantly changed genes

Sample pair	Number of up-	Number of down-	Total number of
(tumor vs normal)	regulated genes1	regulated genes1	changed genes
W904	596	205	801
W361	359	242	601
W380	215	81	296
W904, W361	142	47	189
W904, W380	66	3	69
W361, W380	19	9	28
W904, W361, W380	48	18	66

¹The data is presented as HCC vs normal portion of the liver sample where the normal sample is the baseline.

Table 4 Top 10 up- and down-regulated genes in common on all three tumor/normal sample pairs

Fold change1	Gene title	Gene symbol	Location	Process	Agreement
14.3294	Stearoyl-CoA desaturase (delta-9-desaturase)	SCD	10q23-q24	Fatty Acid Biosynthesis	[7]
11.6297	ELOVL family member 6, elongation of long chain fatty acids (FEN1/Elo2, SUR4/Elo3-like, yeast)	ELOVL6	4q25	Fatty Acid Biosynthesis	[8]
7.7381	Chromosome 9 open reading frame 41	C9orf41	9q21.13		none
5.1036	Cytochrome P450, family 51, subfamily A, polypeptide 1	CYP51A1	7q21.2-q21.3	Electron Transport/ Cholesterol Biosynthesis	[9]
4.1746	H2A histone family, member Z	H2AFZ	4q24	DNA Replication	[10]
3.4227	H2A histone family, member Z	H2AFZ	4q24	DNA Replication	[10]
3.3977	TATA box-binding protein-like protein 1, TBP-like 1	TBPL1	6q22.1-q22.3	Transcription	none
2.9307	Diacylglycerol O-acyltransferase homolog 2 (mouse)	DGAT2	11q13.5	Triacylglycerol biosynthesis	[11]
2.8134	ARP2 actin-related protein 2 homolog (yeast)	ACTR2	2p14	Protein Binding/Structural Molecule Activity	[12,13]
2.8134	Calcium binding protein P22	CHP	15q13.3	Potassium Ion Transport/Small GTPase Mediated Signal Transduction	[14]
-3.6151	Metallothionein 1H	MT1H	16q13	Metal Ion Binding	[15,16]
-3.6353	Metallothionein 2A	MT2A	16q13	Metal Ion Binding	[15,16]
-3.9071	Metallothionein 1X	MT1X	16q13	Metal Ion Binding	[15,17]
-4.1870	EST similar to early growth response 1				[18,19]
-4.5774	Metallothionein 1F (functional)	MT1F	16q13	Metal Ion Binding	[15,16]
-4.9625	Early growth response 1	EGR1	5q31.1	Regulation of Transcription	[18,19]
-5.5956	Metallothionein 1F (functional)	MT1F	16q13	Metal Ion Binding	[15,16]
-5.7266	Sprouty homolog 2 (Drosophila)	SPRY2	13q31.1	Cell-Cell Signaling/ Development/ Organogenesis/ Regulation of Signal Transduction	[20]
-10.3842	Early growth response 1	EGR1	5q31.1	Regulation of Transcription	[18,19]
-24.2139	Insulin-like growth factor binding protein 2, 36 kDa	IGFBP2	2q33-q34	Regulation of Cell Growth	[21]

¹This is the average fold change for all 3 HCC vs normal sample pairs.

Table 5 RT-PCR and microarray results

Gene	Process	W904		W361		W380
		RT-PCR	Microarray	RT-PCR	Microarray	RT-PCR	Microarray
Testis enhanced gene transcript (BAX inhibitor 1) (TEGT)	Negative regulation of apoptosis	No change	Up	Up	Up	Up	Up
Spermidine/spermine N1-acetyltransferase (SSAT)	Polyamine metabolism	Down	Down	Down	Down	No change	Down
Acetyl-CoA synthetase 2 (thiokinase) (ACAS2)1	Metabolism, lipid biosynthesis	Up	Up	Up	Up	Up	No change
Alpha-fetoprotein (AFP)	Immune response, transport	Up	No change	Up	No change	Up	Up
Isocitrate dehydrogenase 3 (NAD+) beta (IDH3B)	Tricarboxylic acid cycle	Up	Up	Undetermined	No change	Up	Up
Carnitine palmitoyltransferase II (CPT2)	Lipid metabolism, fatty acid metabolism, transport	Up	No change	Up	No change	No change	No change
N-myc 2 (NMYC2)	Unknown	Undetermined	N/A	Up	N/A	Undetermined	N/A
C-myc (CMYC)2	Regulation of transcription, DNA-dependent	Up	Up	Down	Up	No change	No change
Fatty-acid-Coenzyme A ligase, very long-chain 1 (FACVL1)	Lipid metabolism, fatty acid metabolism	Down	Down	Undetermined	Down	Up	No change
Hexokinase (GCKR)3	Carbohydrate metabolism	Undetermined	No change	Undetermined	Up	Up	No change

¹RT-PCR results are for ACAS2, microarray results are for a hypothetical protein similar to ACAS2.

²RT-PCR results are for c-myc, microarray results are for a c-myc binding protein and a downstream target.

³RT-PCR results are for glucokinase regulatory protein, microarray results are for an EST similar to hexokinase domain containing 1.