Quantitative Assessment of DNA Methyltransferases in Endometrial Cancer

The qRT-PCR analyses were performed using the SuperScript VILO cDNA Synthesis Kit (Life Technologies) with iTaq Universal SYBR Green Supermix (Bio-Rad). 500 ng total RNA was used in a 20 ul reverse transcription reaction. The cDNA obtained was diluted to a total volume of 100 ul and stored at -20°C. The primers for human DNMT1, DNMT3A, DNMT3B[108 (link)], DNMT2[109 (link)], XIST[110 (link)] and candidate human housekeeping gene 18S rRNA [111 (link)] were used for amplification of the target genes in normal endometrium and endometrial cancer tissues. All primers were synthesized by Integrated DNA Technologies. The qRT-PCR was performed in a 20 ul reaction consisting of 2 ul diluted cDNA, 0.2 uM of each primer and 10 ul iTaq Universal SYBR Green Supermix. All amplifications were carried out in a Bio-Rad CFX96 Real-Time PCR Detection (Bio-Rad) with denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. A melting curve analysis was performed for each run to confirm the specificity of amplification and lack of primer dimers. The qRT-PCR experiments were always run in triplicate. The relative mRNA expression levels of target genes were quantified using the 2-ΔΔCT equation for endometrial normal and cancer tissues. Mean CT of normal endometrium was used as the calibrator sample.