This video demonstrates the procedure of bisulfite modification of genomic DNA from gastrointestinal cancer samples. The technique is beneficial for investigating the relationship between genes’ methylation patterns and establishing their contribution to carcinogenesis.
Protocol
1. Bisulfite Treatment Use 45 µL of the digested tissue solution as the sample. Perform the bisulfite treatment using reagents in a bisulfite conversion kit according to the manufacturer’s instructions (see Table of Materials). Add 5 µL of dilution buffer to the DNA sample and incubate at 37 °C for 15 min (see Table of Materials). While the samples are incubating, prepare the bisulfite conversion reagent b…
Divulgations
The authors have nothing to disclose.
Materials
(NH4)2SO4
Sigma-Aldrich
14148
10% Sodium dodecyl sulfate (SDS)
351-032-721 EA
100% Ethanol
Sigma-Aldrich
24194
ABI StepOnePlus Real-Time PCR System
Applied BioSystems
4376600
96-well Real-Time PCR instrument
CT conversion reagent
Zymo Research
D5001-1
Deoxynucleotide triphosphate (dNTP)
Invitrogen
10297-018
DEPC-treated water
DEPC-treated water
351-068-131
Ethylenediaminetetraacetic acid (EDTA)
Corning
46-034-CL
EZ DNA Methylation Kit
Zymo Research
D5002
Human genomic DNA
New England Bio Labs
N4002S
Infinium Human Methylation 450 assay
Illumina
WG-314
Array platform for complex evaluation of DNA methylation to assess the methylation status of >450,000 CpG sites in the genome
Bisulfite Conversion of Genomic DNA: A Method to Study DNA Methylation in Genomic DNA Samples from Gastrointestinal Cancer Tissue. J. Vis. Exp. (Pending Publication), e20410, doi: (2023).