Optimization of Reference Genes for Normalization of the Quantitative Polymerase Chain Reaction in Tissue Samples of Gastric Cancer


For an exact comparison of mRNA transcription in different samples or tissues with real time quantitativereverse transcription-polymerase chain reaction (qRT-PCR), it is crucial to select a suitable internal referencegene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) have been frequentlyconsidered as house-keeping genes to normalize for changes in specific gene expression. However, it has beenreported that these genes are unsuitable references in some cases, because their transcription is significantlyvariable under particular experimental conditions and among tissues. The present study was aimed to investigatewhich reference genes are most suitable for the study of gastric cancer tissues using qRT-PCR. 50 pairs ofgastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer. AbsoluteqRT-PCR was employed to detect the expression of GAPDH, ACTB, RPII and 18sRNA in the gastric cancersamples. Comparing gastric cancer with corresponding peritumoral tissues, GAPDH, ACTB and RPII wereobviously up-regulated 6.49, 5.0 and 3.68 fold, respectively. Yet 18sRNA had no obvious expression change ingastric cancer tissues and the corresponding peritumoral tissues. The expression of GAPDH, β-actin, RPII and18sRNA showed no obvious changes in normal gastric epithelial cells compared with gastric cancer cell lines.The carcinoembryonic antigen (CEA), a widely used clinical tumor marker, was used as a validation gene.Only when 18sRNA was used as the normalizing gene was CEA obviously elevated in gastric cancer tissuescompared with peritumoral tissues. Our data show that 18sRNA is stably expressed in gastric cancer samplesand corresponding peritumoral tissues. These observations confirm that there is no universal reference gene andunderline the importance of specific optimization of potential reference genes for any experimental condition.