Encyclopedia of Experiments: Cancer Research
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Bisulfite Conversion of Genomic DNA - ConceptTranscript
Perform the bisulfite treatment on 45 microliters of the digested tissue using a bisulfite conversion kit according to the manufacturer's instructions. Add 5 microliters of dilution buffer to the sample and incubate it at 37 degrees Celsius for 15 minutes. Meanwhile, prepare the bisulfite conversion reagent by adding 750 microliters of distilled water and 210 microliters of dilution buffer to one tube of CT conversion reagent. Mix the tubes by vortexing for 10 minutes. Then, add 100 microliters of the prepared CT conversion reagent to each sample and mix by inversion.
Incubate the samples in the dark at 50 degrees Celsius for 12 to 16 hours. After the incubation, place the samples on ice for 10 minutes. Add 400 microliters of binding buffer and mix each sample by pipetting up and down. Load each sample into a spin column and place the column into a 2-milliliter collection tube. Centrifuge the samples at full speed for 1 minute and discard the flow through. Add 200 microliters of wash buffer to each column and spin at full speed for 1 minute, then discard the flow-through.
Add 200 microliters of desulfonation buffer to each column and allow the columns to stand at room temperature for 15 minutes. After the incubation, spin the columns at full speed for 1 minute and discard the flow through. Wash the column twice with 200 microliters of wash buffer, centrifuging for 1 minute at full speed after each wash.
Add 46 microliters of distilled water to each column and place it in a new sterile 1.5-milliliter single-use polypropylene tube. Spin the tubes for 2 minutes to elute the DNA and discard the column. The DNA is now ready for the analysis. Use the bisulfite modified DNA as a template for quantitative methylation-specific PCR to evaluate methylation of the promoter region in each gene analysis. Combine the reagents as described in the text manuscript, and use a 96-well real-time PCR instrument to run the thermal cycling protocol.
