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

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.

Validation of Reference Genes for Normalization of Relative qRT-PCR Studies in Papillary Thyroid Carcinoma

“…After applying the inclusion criteria (Fig. 4) 34 articles were included in the study 21–54 . According to the usage rate, 11 candidate reference genes were selected for validation in the study.…”

Quantitative Proteomic Approach Targeted to Fibrinogen β Chain in Tissue Gastric Carcinoma

“…Antibody-bound proteins were detected by enhanced chemiluminescence using the Chemidoc system after incubation with ECL HRP-conjugated secondary antibodies (1:10,000 dilution, Santa-Cruz, CA, USA) and reaction with ClarityTM Western ECL Substrate (Bio-Rad, Milan, Italy). Because of the lack of universal house-keeping genes to be used as sample loading control in GC [ 62 ], image of the stain-free gel acquired before its transfer was used as control for equal protein loading among samples.…”

Differential Expression of α-Enolase in Clinical Gastric Tissues and Cultured Normal/Cancer Cells in Response to Helicobacter pylori Infection and cagA Transfection

“…To confirm H. pylori infection, the present study adopted PCR method for a high sensitivity [ 18 ]. In addition, as reported, GAPDH, ACTB, RP II and 18S ribosomal RNA (rRNA) are commonly used as the internal control genes in qPCR tests [ 19 ]. While the GAPDH, ACTB and RP II expressions may change with the severity of gastric pathological lesions, the 18S ribosomal RNA can be kept at a relatively stable level [ 19 ].…”