Optimizing a test for TWIST2 gene methylation detection based on MethylScreen technology

Abstract

TWIST2 is a transcription factor that regulates events essential for embryogenesis, tumor development, and metastasis. Studies have identified DNA methylation as one of the epigenetic changes responsible for TWIST2 gene expression regulation, so it is important to study TWIST2 methylation changes in cancer cells. Analyzing DNA methylation by conventional methods is a time and effort consuming procedure.

In this study, we designed an assay and primers to study the methylation of 14 CpG sites in the TWIST2 gene based on MethylScreen technology. We also optimized the detection reaction conditions and tested the validity of this assay in ovarian cancer SK-OV-3 and A2780 cell lines. Gene expression changes in the two cell lines were also studied by qPCR.

Our results indicated that the designed primers give a single 151 bp amplicon. The optimal annealing temperature of these primers is 60 °C. Tested primers were able to amplify 50ng of DNA in the early qPCR reaction. The results also showed that the TWIST2 gene was not methylated in SK-OV-3 cells, While the percentage of methylation of the studied region increased in A2780 cells, it was 3.7% hypermethylated, 88.5% intermediately methylated, and 7.8% unmethylated. Increased Methylation in A2780 cells was associated with a 0.05-fold decrease in gene expression when compared to SK-OV-3 cells.

We conclude that the designed test can be used to determine the methylation of the TWIST2 gene, in a relatively short period of time and at a lower cost compared to the traditional methods.