Epigenetic alterations such as DNA methylation not only regulate mammalian gene expression during developmental stages but also play a key role in tumor progression. Hypermethylation or aberrant methylation of promoters causes transcriptional repression of critical growth regulators such as tumor suppressor genes, facilitating neoplastic transformation. Such transcriptional silencing is associated with abnormal methylation of DNA at certain CpG islands that lie in the promoter regions of these genes. The study of molecular events underlying alterations in DNA methylation patterns could lead to the development of novel therapies based on hypomethylating agents which reverse gene silencing and inhibit oncogenesis. However, the study of gene-promoter-specific hypermethylation patterns in cancer has proven to be an arduous process because of the technical difficulties involving the analysis of DNA methylation.
Conventional molecular biology methods to examine methylation patterns of DNA include the use of methylation-sensitive restriction enzymes followed by Southern blot analysis of the digested DNA, or sodium bisulfate conversion of methylated bases followed by PCR and sequencing of the PCR amplification product. Both these methods are extremely tedious, prone to errors and allow analyses of a limited number of promoters in a single experiment. The biggest advantage of the Panomics TranSignal™ Promoter Methylation Arrays is its ease of use. The array kit is based on high-throughput screening of promoter methylation patterns. Briefly, genomic DNA is digested with a restriction enzyme to produce small fragments of methylated DNA. The digests are purified and annealed to linkers and subsequently incubated with MBP (methylation binding protein) to form protein-DNA complexes. These complexes are isolated, purified and subsequently labeled with biotin-dCTP. In the final step is hybridization of the labeled DNA on the array. The signals are detected by using Strepavidin-HRP, which eliminates the use of hazardous radioactive chemicals associated with traditional Southern blotting techniques. A single experiment using the Panomics array takes two days to complete and permits analysis of 82 different promoter regions from each sample. The data obtained from these arrays are very high quality, consistent and extremely reproducible. The user manual provided with the kit provides the detailed experimental procedure and is easy to understand. Furthermore, the technical service of Panomics is excellent.
The focus of our laboratory is to study the differential gene expression patterns of tumor suppressor genes in response to growth factors or apoptotic agents. We wanted to examine whether treatment of non-small cell lung cancer cells with the anti-cancer drug gemcitabine induced alterations in the DNA methylation patterns of these cells. We extracted genomic DNA from A549 NSCLC cells which were treated with 20µM gemcitabine using DNeasy Tissue Kit (Qiagen). Genomic DNA from untreated A549 served as the control for the experiment. Upon completion of the experiment, the chemiluminiscent signals on the array were visualized by autoradiography. The position of the spots on the X-ray film were matched to the key of the array (which is provided in the technical manual) to determine which specific genes has been methylated.
The disadvantage of the kit is that it is extremely expensive and may not be affordable for all laboratories. The kit contains 3 arrays and the reagents provided are enough for a single experiment. Although the arrays are re-usable, you have to order a refill kit to use them again and the cost of the refill kit is marginally lower than a brand new kit. Although the technical instructions with the kit are excellent, certain key details are missing. For example, the manual provides detailed instructions about how to strip the arrays but does not say whether the stripped arrays should be kept at room temperature or 4oC for long-term storage. We dried the arrays at 80oC and stored them at room temperature. The arrays worked if we re-used them once, however, when used more than once, they started giving background signal. Another disadvantage of the kit is that although the key to the array gives the identity of the promoters, no accession numbers or sequences are provided. One of the genes in the array is listed as IFN (interferon), however, it is not mentioned which member of the interferon family is being referred to, a problem further compounded by the absence of a sequence and accession number. However, the advantages of the Panomics TranSignal™ Promoter Methylation Arrays far outweigh the minor disadvantages and it is an invaluable asset for researchers involved in the study of DNA methylation in mammalian cells.
Piyali Dasgupta
Postdoctoral Fellow
H. Lee Moffitt Cancer Center
Drug Discovery