Abstract
BackgroundHousekeeping genes are routinely used as endogenous references to account for experimental differences in gene expression assays. However, recent reports show that they could be de-regulated in different diseases, model animals, or even under varied experimental conditions, which may lead to unreliable results and consequently misinterpretations. This study focused on the selection of suitable reference genes for quantitative PCR in human hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) with different clinical outcomes.MethodsWe evaluated 6 commonly used housekeeping genes' expression levels in 108 HBV-related HCCs' matched tumor and non-tomor tissue samples with different clinical outcomes and 26 normal liver specimens by real-time PCR. The expression stability of the 6 genes was compared using the software programs geNorm and NormFinder. To show the impact of reference genes on data analysis, we took PGK1 as a target gene normalized by each reference gene, and performed one-way ANOVA and the equivalence test.ResultsWith the geNorm and NormFinder software programs, analysis of TBP and HPRT1 showed the best stability in all tissue samples, while 18s and ACTB were less stable. When 18s or ACTB was used for normalization, no significant difference of PGK1 expression (p > 0.05) was found among HCC tissues with and without metastasis, and normal liver specimens; however, dramatically differences (p < 0.001) were observed when either TBP or the combination of TBP and HPRT1 were selected as reference genes.ConclusionTBP and HPRT1 are the most reliable reference genes for q-PCR normalization in HBV-related HCC specimens. However, the well-used ACTB and 18S are not suitable, which actually lead to the misinterpretation of the results in gene expression analysis.
Highlights
Housekeeping genes are routinely used as endogenous references to account for experimental differences in gene expression assays
Typical housekeeping genes (HKGs) including glyceraldehydes 3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), TATA-binding protein (TBP), 18S-r RNA (18S) ribosomal RNA (18S) and many more have often been adopted from the literatures as reference genes without taking into account their specific tissue dependent behavior or the special design of the respective study [6,9,10,11,12,13,14,15,16]
We evaluated 69 articles that had used various reference genes, and found that betaactin (ACTB; 25 times; 36%), glyceraldehydes-3-phosphate dehydrogenase (GAPDH; 19 times; 28%), 18S-r RNA (18S; 12 times; 17%), TATA box binding protein (TBP; 5 times; 7%) and Hypoxanthine phosphoribosyltransferase I (HPRT1; 4 times; 6%) and ribosomal protein L 13a (RPL13A; 4 times; 6%) were commonly used (Table 1)
Summary
Housekeeping genes are routinely used as endogenous references to account for experimental differences in gene expression assays. Recent reports show that they could be de-regulated in different diseases, model animals, or even under varied experimental conditions, which may lead to unreliable results and misinterpretations. Nowadays, housekeeping genes (HKGs) are routinely-used as references in qPCR to normalize experimental data, such as differences in RNA quantity and quality, the overall transcriptional activity and differences in the cDNA synthesis [4], because, theoretically, HKGs are supposed to exhibit consistent, nonregulated, stable expression among different space-time and different tissues, even intervention models [5,6]. Being de-regulated in various samples those so-called HKGs for qPCR normalization on cancer research may lead to unreliable results and misinterpretation [13,15,17]. It is crucial to find appropriate reference genes for qPCR normalization on specific cases
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