With the usage of RT-qPCR, we researched the expression of seven genes (ACTB, B2M, G6PD, GAPD, GUSB, HPRT and PPIA) as reliable reference genes in eutopic and ectopic endometrial tissue specimens obtained during standard surgery of women of reproductive age. Stability of expression level was analyzed by the most universal MS Excel plug-ins including: geNorm, NormFinder and BestKeeper. The descriptive statistics were evaluated using Statistica software.
The distribution of threshold (Ct) values was not equal. We identified genes with higher expression level (referring to Ct values) such as ACTB and B2M, medium e.g., GAPD and low expression level, e.g., G6PD and HPRT. We demonstrated that the stability of the analyzed reference genes was not homogenous, and different algorithms pointed to PPIA, GAPD and B2M as the most stable ones in eutopic and ectopic endometrium. On the contrary to these, GUSB and G6PD were the most unstable ones.
In RT-qPCR-based analyses of gene expression level in eutopic and ectopic endometrium, we strongly recommend that a minimum of two reference genes are to be used and we determined that the most suitable seem to be PPIA and GAPD.