Aberrant DNA methylation patterns have been implicated in many disease systems, including cancer. Bisulfite sequencing is the gold standard in assessing the methylation status of a particular region because only sequencing provides direct detection of methylation events, as well as information across the entire amplified region. When working with clinical samples, researchers often need to use DNA that has been stored as formaldehyde-fixed paraffin-embedded (FFPE) tissue blocks. DNA from FFPE tissue blocks is difficult to analyze due to varying degrees of fragmentation and protein contamination. This protocol describes how easy it is to generate high quality methylation results from DNA isolated from FFPE samples.
STEP 1 DNA Isolation from FFPE Tissues
DNA is isolated using the RecoverAll™ Total Nucleic Acid Isolation Kit, which provides high quality, high-molecular-weight DNA, free from PCR inhibitors. RecoverAll kits are optimized to release a maximal amount of DNA fragments of all sizes, with typical yields of more than 50% that of DNA recovered from unfixed tissue from the same sample source. STEP 2 Bisulfite Conversion
DNA is converted using the methylSEQr™ Bisulfite Conversion Kit. This kit uses size exclusion column purification, which increases sample yields and reduces the amount of bias seen with resin purification methods. Unbiased DNA recovery of methylated and unmethylated fragments enables more accurate downstream quantitative analysis. The methylSEQr kit uses gentle denaturation conditions that reduce DNA fragmentation and increase the amount of high quality DNA available for amplification. STEP 3 Assay Design and Amplification
Primers are designed using Methyl Primer Express® Software v1.0, a free online primer design tool specifically for methylation studies. The software performs an in silico bisulfite conversion (Cs are converted to Ts) and aids in the selection of primers.
Amplification of the bisulfite-converted DNA from step 2 is performed using AmpliTaq Gold® DNA Polymerase on Applied Biosystems thermal cyclers, including the Veriti™ 96-Well Fast Thermal Cycler (in standard ramp mode). The PCR product is purified enzymatically with ExoSAPIT® Reagent (USB Corporation). STEP 4 Detection and Analysis
Amplified products from step 3 are sequenced with the BigDye® Terminator v1.1 Cycle Sequencing Kit and M13-tailed primers, using a slightly modified thermal cycler profile. Bisulfite sequencing reactions are purified using the BigDye XTerminator™ Purification Kit, which removes unincorporated BigDye Terminators quickly and easily. The reactions are then run on a 3130 Genetic Analyzer. A Complete Solution
Applied Biosystems provides a streamlined workflow for bisulfite sequencing that takes you from DNA isolation to final results, with optimized protocols for each step.
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