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

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

1. Use 45 µL of the digested tissue solution as the sample.
2. Perform the bisulfite treatment using reagents in a bisulfite conversion kit according to the manufacturer’s instructions (see Table of Materials).
   1. Add 5 µL of dilution buffer to the DNA sample and incubate at 37 °C for 15 min (see Table of Materials).
   2. While the samples are incubating, prepare the bisulfite conversion reagent by adding 750 µL of dH₂O and 210 µL of dilution buffer to one tube of CT conversion reagent (see Table of Materials

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(NH4)2SO4	Sigma-Aldrich	14148
10% Sodium dodecyl sulfate (SDS)	Quality Biological	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