Bisulfite Conversion of Genomic DNA: A Method to Study DNA Methylation in Genomic DNA Samples from Gastrointestinal Cancer Tissue

Published: April 30, 2023

Abstract

Source: Bonetto, A. et al. The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia. J. Vis. Exp. (2016)

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…

Divulgaciones

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
Light Cycler 480 Roche 5015278001
M-Binding Buffer Zymo Research D5002-3
M-Desulphonation Buffer Zymo Research D5002-5
M-Dilution Buffer Zymo Research D5002-2
M-Wash Buffer Zymo Research D5002-4
Proteinase K New England Biolabs P8107S
Single-use polypropylene (Eppendorf) tube Eppendorf 24533495
Tris hydrochloride (Tris-HCL) 2 M pH 8.8 Quality Biological 351-092-101
Zymo Spin 1 Column Zymo Research C1003
β-Mercaptoethanol Sigma-Aldrich M3148
Fluorescein Bio-Rad #1708780
Platinum Taq polymerase ThermoFisher Scientific 10966-034

Tags

Play Video

Citar este artículo
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).

View Video