Selection of reference genes for RT-qPCR analysis in tumor tissues from male hepatocellular carcinoma patients with hepatitis B infection and cirrhosis.
BACKGROUND
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has been widely used to quantify relative gene expression because of the high specificity, sensitivity and accuracy of this technique. However, its reliability is strongly depends on the expression stability of reference gene used for data normalization. Therefore, identification of reliable and condition specific reference genes is critical for the success of RT-qPCR.


OBJECTIVE
Hepatitis B virus (HBV) infection, male gender and the presence of cirrhosis are widely recognized as the leading independent risk factors for the development of hepatocellular carcinoma (HCC). This study aimed to select reliable reference gene for RT-qPCR analysis in HCC patients with all of those risk factors.


METHODS
Six candidate reference genes were analyzed in 33 paired tumor and non-tumor tissues from untreated HCC patients. The genes expression stabilities were assessed by geNorm and NormFinder.


RESULTS
C-terminal binding protein 1(CTBP1) was the most stable gene among the 6 candidate genes evaluated by both geNorm and NormFinder. The expression stability values were 0.08 for CTBP1 and UBC, 0.09 for HPRT1, 0.12 for HMBS, 0.14 for GAPDH and 0.18 for 18S with geNorm analysis. The stability values suggested by NormFinder software were CTBP1: 0.044, UBC: 0.063, HMBS: 0.072, HPRT1: 0.072, GAPDH: 0.098 and 18S rRNA: 0.161.


CONCLUSIONS
This is the first systematic analysis which suggested CTBP1 as the highest expression-stable gene in human male HBV infection related-HCC with cirrhosis. We recommend CTBP1 as the best candidate reference gene when RT-qPCR was used to determine gene(s) expression in HCC. This may facilitate the relevant HBV related HCC studies in the future.
Beijing Artificial Liver Treatment and Training Center, Beijing Youan Hospital, Capital Medical University, Beijing, China.
Shuang
S
Liu
Department of Microbiology and Infectious Disease Center, Peking University Health Science Center, Beijing, China.
Pengfei
P
Zhu
Henan Cancer Hospital, Zhengzhou, Henan, China.
Ling
L
Zhang
Department of Microbiology and Infectious Disease Center, Peking University Health Science Center, Beijing, China.
Shanlong
S
Ding
Beijing Artificial Liver Treatment and Training Center, Beijing Youan Hospital, Capital Medical University, Beijing, China.
Sujun
S
Zheng
Beijing Artificial Liver Treatment and Training Center, Beijing Youan Hospital, Capital Medical University, Beijing, China.
Yang
Y
Wang
Department of Microbiology and Infectious Disease Center, Peking University Health Science Center, Beijing, China.
Fengmin
F
Lu
D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D020869:Gene Expression Profiling / Q000592:standards; D019694:Hepatitis B, Chronic / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008103:Liver Cirrhosis / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards; D059467:Transcriptome
D014408:Biomarkers, Tumor
C-terminal binding protein 1; Hepatocellular carcinoma; RT-qPCR; hepatitis B virus; liver cirrhosis; male gender; reference gene