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…
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 |
| 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
Citer Cet Article
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).