Selection of reference genes for quantitative real-time reverse transcription-polymerase chain reaction in concanavalin A-induced hepatitis model.
Quantitative real-time reverse transcription-polymerase chain reaction (Q-PCR) has become an indispensable technique for accurate determination of gene expression in various samples. In mice, intravenous injection of concanavalin A (ConA) leads to acute hepatitis and liver injury. Functional studies based on this model have provided insights for understanding the mechanisms of liver injury. However, no data have been reported to validate reference genes during the progression of ConA-induced hepatitis (CIH). In this study, IkappaBalpha and C/EBPbeta messenger RNA (mRNA) levels were examined using Q-PCR with ACTB as the reference gene after ConA injection. However, we got inconsistent results with previous reports determining IkappaBalpha and C/EBPbeta mRNA expression levels. The results indicate the necessity for stability analysis of candidate reference genes in the CIH model. geNorm, NormFinder, and BestKeeper software analysis indicates that ACTB is the most unstable gene during CIH progression among the 10 reference genes tested, whereas RPLP0 or HPRT1 is the most stable one. This study demonstrates that some of the commonly used reference genes are inadequate for normalization of Q-PCR data due to their expression instability. Furthermore, this study validates HPRT1 and RPLP0 as appropriate reference genes for Q-PCR analysis in the CIH model.
Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao-Tong University School of Medicine, Shanghai 200031, China.
Guojun
G
Shi
Zhijian
Z
Zhang
Dechun
D
Feng
Yan
Y
Xu
Yan
Y
Lu
Jiqiu
J
Wang
Jingjing
J
Jiang
Zhiguo
Z
Zhang
Xiaoying
X
Li
Guang
G
Ning
D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism; D056486:Chemical and Drug Induced Liver Injury / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D003208:Concanavalin A; D004195:Disease Models, Animal; D015870:Gene Expression; D005796:Genes; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000378:metabolism; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*
D022782:CCAAT-Enhancer-Binding Protein-beta; D012333:RNA, Messenger; D003208:Concanavalin A; D051550:I-kappa B Kinase