Abstract
BackgroundReverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results.MethodsWe assessed the suitability of six possible reference genes, beta-actin (ACTB), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), beta-2-microglobulin (B2M), ribosomal subunit L29 (RPL29) and 18S ribosomal RNA (18S rRNA) in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values.ResultsThis RT-qPCR study showed that there are statistically significant (p < 0.05) differences in the expression levels of HPRT1 and 18S rRNA in 'normal-' versus 'tumor stomach tissues'. The stability analyses by geNorm suggest B2M-GAPDH, as best reference gene combination for 'stomach cancer cell lines'; RPL29-HPRT1, for 'all stomach tissues'; and ACTB-18S rRNA, for 'all stomach cell lines and tissues'. NormFinder also identified B2M as the best reference gene for 'stomach cancer cell lines', RPL29-B2M for 'all stomach tissues', and 18S rRNA-ACTB for 'all stomach cell lines and tissues'. The comparisons of normalized expression of the target gene, GPNMB, showed different interpretation of target gene expression depend on best single reference gene or combination.ConclusionThis study validated RPL29 and RPL29-B2M as the best single reference genes and combination, for RT-qPCR analysis of 'all stomach tissues', and B2M and B2M-GAPDH as the best single reference gene and combination, for 'stomach cancer cell lines'. Use of these validated reference genes should provide more exact interpretation of differential gene expressions at transcription level in stomach cancer.
Highlights
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression
In an evaluation of 115 articles published from May 2007 to November 2009, we found that glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (53 cases; 46.1%) and ACTB (41 cases; 35.7%) were the most frequently used reference genes in gastric cancer studies; followed by 18S rRNA (8 cases; 7.0%), beta-2-microglobulin (B2M; 3 cases; 2.6%), hypoxanthine phosphoribosyl transferase 1 (HPRT1; 2cases; 1.7%), TATA binding protein (TBP; 1 case; 0.9%), and beta-tubulin (TUBB; 1 case; 0.9%)
We investigated the five reference genes that have been most frequently used genes in stomach cancer studies (ACTB, GAPDH, B2M, 18S rRNA, and HPRT1) and for comparison, RPL29, a reference gene used in other cancer studies, in 'non-stomach cancer cell lines', 'stomach cancer cell lines', 'normal stomach tissues' and 'tumor stomach tissues' (Table 1)
Summary
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful tool for validating the observed gene expression differences, because of its greater sensitivity and specificity. Several tools for statistical analysis such as NormFinder [7], geNorm [8], BestKeeper [9] have been developed to help in the choice of appropriate reference genes. These tools assess the variations in the expression of a number of potential reference genes and suggest which reference gene(s) is appropriate for normalization of gene expression data in a given study
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