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

Protocolo

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). Mix the tubes by vortexing for 10 min.
   3. After the 15 min incubation, add 100 µL of the prepared CT conversion reagent to each sample and mix by inversion.
   4. Incubate the samples in the dark at 50 °C for 12 to 16 h.
   5. After the incubation, remove the samples and place them on ice for 10 min.
   6. Add 400 µL of binding buffer and mix each sample by pipetting up and down (see Table of Materials). Load each sample into a spin column and place the column into a 2 mL collection tube (see Table of Materials).
   7. Centrifuge each sample at full speed (10,000 x g) for 1 min and discard the flow-through.
   8. Add 200 µL of wash buffer to each column and spin at full speed for 1 min, discarding the flow-through (see Table of Materials).
   9. Add 200 µL of desulfonation buffer to each column and allow the column to stand at room temperature for 15 min (see Table of Materials). After the incubation, spin the columns at full speed for 1 min and discard the flow-through.
   10. Add 200 µL of wash buffer to each column and spin at full speed for 1 min (see Table of Materials).
   11. Repeat this step one more time.
   12. Add 46 µL of dH₂O to each column and place each column in a new sterile 1.5 mL single-use polypropylene tube (see Table of Materials). Spin each tube for 2 min to elute the DNA.
3. Remove each spin column from the single-use polypropylene tube and discard (see Table of Materials). The DNA is now ready for the analysis.

2. Quantitative Methylation-Specific PCR (qMSP)

1. Use the bisulfite-modified DNA from step 1.3 as a template for fluorescence-based real-time PCR in qMSP to evaluate methylation of the promoter region in each gene analysis.
2. Perform qMSP using a 96-well Real-Time PCR instrument (see Table of Materials).
   1. Check for the promoter methylation status of the target gene on the bisulfite-modified DNA using 200 nM forward primer, 200 nM reverse primer, and 80 nM probes. Prepare the master mix with 16.6 mM (NH₄)₂SO₄, 67 mM Tris pH 8.8, 10 mM β-mercaptoethanol, 10 nM fluorescein, 0.166 mM of each deoxynucleotide triphosphate, and 0.04 U/μl of DNA polymerase (see Table of Materials). The final reaction volume in each well for all assays is 25 μL.
   2. Perform cycling of qMSP as follows: 95 °C for 5 mins, followed by 55 cycles of 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min.
      NOTE: Target gene should be chosen based on the criteria of having larger beta values, being related to CpG islands in the promoter region, and being suitable for primer and probe design for qMSP.
3. Use Human genomic treated with CpG Methylase (M.SssI) as a positive methylation control (see Table of Materials).
   NOTE: The final quantification of methylation is defined as the relative methylation value (RMV) and calculated as 2^–ΔΔCt for each methylation detection replicate compared to the mean Ct for β-Actin (ACTB). Primer and probe sequences are shown in Table 1. A Ct of 100 is used for undetected replicates, which gives a value of 2^–ΔΔCt close to zero. The following formula is used:
   mean 2^–ΔΔCt (RMV) = (2^{–ΔΔCt_replicate_1} + 2^{–ΔΔCt_replicate_2} + 2^{–ΔΔCt_replicate_3})/3.

Gene	Forward 5' - 3'	Reverse 5' - 3'	Probe
B-ACTIN	TAG GGA GTA TAT AGG TTG GGG AAG TT	AAC ACA CAA TAA CAA ACA CAA ATT CAC	\56-FAM\ TGT GGG GTG \ZEN\ GTG ATG GAG GAG GTT TAG \3IABkFQ\
EPB41L3	GGG ATA GTG GGG TTG ACG C	ATA AAA ATC CCG ACG AAC GA	AAA TTC GAA AAA CCG CGC GAC GCC GAA ACC A

Materiales

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