Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

AIM:To evaluate the suitability of reference genes in gastric tissue samples and cell lines.METHODS:The suitability of genes ACTB,B2M,GAPDH,RPL29,and 18S rRNA was assessed in21 matched pairs of neoplastic and adjacent nonneoplastic gastric tissues from patients with gastric adenocarcinoma,27 normal gastric tissues from patients without cancer,and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR).The ranking of the best single and combination of reference genes was determined by NormFinder,geNorm,BestKeeper,and DataAssist.In addition,GenEx software was used to determine the optimal number of reference genes.To validate the results,the mRNA expression of a target gene,DNMT1,was quantified using the different reference gene combinations suggested by the various software packages for normalization.RESULTS:ACTB was the best reference gene for all gastric tissues,cell lines and all gastric tissues plus cell lines.GAPDH+B2M or ACTB+B2M was the best combination of reference genes for all the gastric tissues.On the other hand,ACTB+B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines.According to the GenEx software,2 or 3 genes were the optimal number of references genes for all the gastric tissues.The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes.The level of expression of DNMT1 in neoplastic,adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH+B2M(P=0.32),ACTB+B2M(P=0.61),or GAPDH+B2M+ACTB(P=0.44).CONCLUSION:GAPDH+B2M or ACTB+B2M is the best combination of reference gene for all the gastric tissues,and ACTB+B2M is the best combination for the cell lines tested.

S53G SNP in Cathepsin B: Can Be Used as a Molecular Biomarker to Breast Cancer?

Cathepsin B (CTS B) is a proteolytic enzyme that participates in several important biological processes. However, when it is altered can be involved in development of breast cancer, a disease with high incidence and mortality rate among women. Many studies have shown a correlation between high CTS B expression and breast tumor. Furthermore, it has been shown that the SNP rs1803250 (S53G) leads to structural and functional changes in protein that can make it pathogenic. The present study aimed to evaluate a possible association of SNP rs1803250 (S53G) with breast cancer. For this, real-time PCR was performed on a sample collected in the State of Pará, with 127 patients and 122 controls. The SNP frequency in this region was 0.12, according to a research project in progress that aims to identify Amerindians molecular alterations. This indicates that the SNP is found in region with a distribution close to worldwide frequency of 0.09. Our results showed that the SNP was in Hardy-Weinberg Equilibrium in collected sample, but C variant allelic frequency was 0.08 in both patients and control groups, which is extremely similar to global average. Moreover, homozygotes CC was not found in the sample and SNP genotypes frequency in patients and control groups was not significantly different. In addition, statistical analysis showed that the SNP did not have to correlate with tumor subtypes nor with tumor staging. Therefore, according to the analyzed sample, the SNP rs1803250 has no association with breast cancer and it cannot be considered a molecular biomarker for breast cancer.