Abstract
For an exact comparison of mRNA transcription in different samples or tissues with real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), it is crucial to select a suitable internal reference gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) have been frequently considered as house-keeping genes to normalize for changes in specific gene expression. However, it has been reported that these genes are unsuitable references in some cases, because their transcription is significantly variable under particular experimental conditions and among tissues. The present study was aimed to investigate which reference genes are most suitable for the study of gastric cancer tissues using qRT-PCR. 50 pairs of gastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer. Absolute qRT-PCR was employed to detect the expression of GAPDH, ACTB, RPII and 18sRNA in the gastric cancer samples. Comparing gastric cancer with corresponding peritumoral tissues, GAPDH, ACTB and RPII were obviously up-regulated 6.49, 5.0 and 3.68 fold, respectively. Yet 18sRNA had no obvious expression change in gastric cancer tissues and the corresponding peritumoral tissues. The expression of GAPDH, β-actin, RPII and 18sRNA 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 tissues compared with peritumoral tissues. Our data show that 18sRNA is stably expressed in gastric cancer samples and corresponding peritumoral tissues. These observations confirm that there is no universal reference gene and underline the importance of specific optimization of potential reference genes for any experimental condition.
Highlights
With the unique features of accurate quantification, high sensitivity and throughput (Huggett et al, 2005; Radonić et al, 2004), the technique of real time quantitative reverse transcription-polymerase chain reaction has been applied extensively for detection of specific gene expression
The present study was aimed to investigate which reference genes are most suitable for the study of gastric cancer tissues using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). 50 pairs of gastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer
After qRT-PCR, in gastric cancer tissues, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was upregulated by 2.7±0.41 cycles (6.49±1.32folds), Beta-actin up-regulated by 2.33±0.32cycles (5.0±1.24folds), RNA polymerase II (RPII) up-regulated by 1.88±0.21cycles (3.68±1.15 folds)
Summary
With the unique features of accurate quantification, high sensitivity and throughput (Huggett et al, 2005; Radonić et al, 2004), the technique of real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been applied extensively for detection of specific gene expression. While performing qRT-PCR experiments, a reference gene, called internal standard gene or normalized gene is required to calibrate the expression level of target gene to obtain objective and trustworthy results. This method is based on the assumption that the expression level of the normalizing gene does not change from sample to sample. As demonstrated by the results of many experiments, a stable expression of these internal reference genes was only detectable in some particular tissues and cells or under some special experimental conditions.
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