MethylDetector™ Kit From Active Motif

Epigenetic modification of CpG islands (CGIs) in promoter regions is an important regulatory mechanism of gene expression in eukaryotic cells. Hypermethylation of CGIs may silence a gene, whereas hypomethylation of previously methylated CGIs allows gene expression. The pattern of methylation is cell-type-specific and established during development of organism. Several convergent studies have established that profound and widespread alterations of DNA methylation contribute to the pathophysiology of cancer as well as the aging process. The detection and quantification of DNA methylation alterations is likely a promising tool for diagnosis of many types of cancer. In addition, potential reversibility of epigenetic changes in genes involved in tumor progression makes them attractive therapeutic targets.

The process of DNA methylation involves addition of a methyl group to the fifth carbon of cytosine in a CpG dinucleotide by DNA methyltransferase. The bisulfite conversion method is one of the well-established techniques to study DNA methylation. However, the protocol for bisulfite conversion is technically challenging, laborious and its optimization takes time. The MethylDetector™ Kit provides an optimized set-up for performing the bisulfite conversion reaction. There is no need for the initial alkaline denaturation of DNA in the first step, which saves valuable time and reduces errors.

During the course of our studies, we wanted to examine whether treatment of A549 non-small cell lung cancer cells with cisplatin produced any changes in the methylation pattern of genes involved in angiogenesis and metastasis. We treated A549 cells with 20 M cisplatin for 12 hours and then extracted genomic DNA from both the control and untreated A549 cells. The next step was to perform the conversion reaction using the MethylDetector™ Kit. The kit provides optimized reagents and buffers and a simple, yet detailed protocol. The technical instruction booklet is excellent. The purification and desulfonation of DNA was done on easy-to use columns. This improves the efficiency of DNA recovery after the conversion reaction. Subsequently, the DNA is amplified by PCR or nested PCR, for analysis.

The biggest advantage of this kit is that the positive control primer pairs provided are specific for bisulfite converted DNA, so there is no background from non-converted DNA. These positive control PCR primers amplify the p16 locus, whose demethylation has been implicated in the progression of several cancers. The kit contains reagents for 50 reactions and is very reasonably priced – another major plus for many research labs.

The only negative side to the kit is that the shelf life of the reagents and buffers is not too long. Most of the reagents are stable for about 6 months. However, once you dilute the reagents to set up the reactions, the shelf life of these diluted reagents is about a week. The concentrated reagents are supplied in 5 batches and each batch is designed for 10 reactions. So the best strategy is to prepare at least 10 samples in advance and then analyze all the samples in a one or two runs of the kit. I would also recommend that the conversion reaction be done at least in duplicate to ensure a high degree of reproducibility. However, these drawbacks are extremely minor and I would wholeheartedly recommend Active Motif’s MethylDetector™ Kit for any laboratory studying epigenetic modifications of DNA in disease.