Abstract: This study had evaluated three commercially available bisulfite modification kits with regard to the procedural DNA loss and the efficacy of cytosine conversion to uracil. The efficacy of DNA conversion was estimated by pyrosequencing of eight CpG sites of unmethylated and fully enzymatically methylated templates of RARβ gene promoter region and cirDNAs of healthy donors and prostate cancer patients. The procedural DNA loss was calculated by methyl-independent qPCR for ubiquitously distributed L1RE1 retrotransposable elements. Our data demonstrates that all commercial kits displayed similar conversion efficacy of the unmethylated cytosines close to 99% independently of DNA concentration. However, when low DNA concentrations were used, the observed basic level of genomic DNA methylation increased to 11% depending on the position of CpG site. Qiagen and Chemicon kits recovered no more than 20% starting material at a high DNA input (500 ng/probe) and only 2.7–5.8% for low DNA input (10 ng/probe). EZ DNA Methylation-Gold Kit from Zymo Research provided the highest recovery regardless of the initial DNA input with average rates of no less than 86%. These results suggest that the EZ DNA Methylation-Gold Kit is the most appropriate tool for bisulfite modification of cirDNA when assaying DNA in low amounts.Abstract: This study had evaluated three commercially available bisulfite modification kits with regard to the procedural DNA loss and the efficacy of cytosine conversion to uracil. The efficacy of DNA conversion was estimated by pyrosequencing of eight CpG sites of unmethylated and fully enzymatically methylated templates of RARβ gene promoter region and ci...Show More
