5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

Overview

This video describes the technique of dot-blot to determine the level of DNA methylation in human chondrocytes in vitro via the blotting of DNA samples on a nylon membrane and the subsequent visualization using monoclonal antibodies. This helps to determine the chondrocytes' phenotype at various stages in culture.

Protocol

1. Human Articular Cartilage Tissue Collection and Chondrocyte Culture

1. Preparation of materials
   1. Prepare Dulbecco's modified eagle medium (DMEM) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Prepare 1 mg/mL collagenase II, 0.25% trypsin-EDTA, phosphate-buffered saline (PBS), and a cell strainer (40 µm nylon).
2. Human articular cartilage tissue collection
   1. Isolate articular cartilage from the knee joints of donor patients after trauma. Obtain informed consent from all participants.
   2. Dice the cartilage into 1-2 mm³ pieces using a sterile scalpel and digest chondrocytes from the minced cartilage with 1 mg/mL collagenase II in DMEM at 37 °C for 12-16 h.
   3. Filter the resulting cell suspension through a cell strainer (40 µm) and wash twice with PBS.
   4. Count cells with a hemocytometer and seed at a density of 20,000-30,000 cells/cm² in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
   5. Culture in an incubator at 37 °C.
3. Monolayer chondrocyte expansion
   1. Harvest sub-confluent cells using 0.25% trypsin-EDTA and re-plate at a density of 6,600 cells/cm². Change the medium twice a week.
   2. Culture chondrocytes in monolayers for up to six passages and assess at passages 1, 2, 3, 4, and 5.

2. Genomic DNA (gDNA) Extraction

1. Collect cells and resuspend in 500 µL lysis buffer (15 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 0.5% SDS, 200 µg/mL RNase A) per 10 million cells. Resuspend cells by pipetting and rapid inversion, and then incubate for 1 h at 37 °C.
2. Add proteinase K at a concentration of 160 µg/mL of cell lysate and invert the mixture vigorously. Incubate 6 h at 55 °C.
3. Add one volume of Tris pH 7.9 saturated phenol:chloroform:isoamyl alcohol (25:24:1) to the sample. Vortex or shake the sample by hand thoroughly for approximately 20 s.
4. Centrifuge the sample at room temperature for 5 min at 13,000 × g. Remove the upper aqueous phase and transfer the layer to a fresh tube.
5. Extract with an equal volume of chloroform to remove phenol and transfer the top aqueous phase to a fresh tube.
6. Precipitate DNA by adding 0.1 sample volume of 3 M sodium acetate pH 8.0 and 2 volumes of 100% ethanol.
7. Store the tube at -20 °C overnight to precipitate the gDNA.
8. Centrifuge the sample at 4 °C for 10 min at 16,000 x g to pellet gDNA.
9. Wash the sample three times with 70% ethanol, and centrifuge at 4 °C for 2 min at 13,000 x g.
10. Remove the supernatant carefully and then air dry. Resuspend the DNA in 10 mM Tris pH 8.0, 0.1 mM EDTA.

3. DNA Methylation Profiling

1. Use a DNA methylation kit to perform the bisulfite conversion reaction using a total of 500 ng of the genomic DNA, according to the manufacturer's protocol. Elute in 10 µL of elution buffer (50 ng/µL).
2. Perform the DNA methylation profiling using a commercial kit according to the manufacturer's protocol.

4. Dot Blot Analysis

1. Denature the isolated DNA (1 mg per sample) in 0.1 M NaOH for 10 min at 95 °C. Neutralize the DNA with 1 M NH₄OAc on ice, and then dilute two-fold. Spot 2 µL of the serial diluted genomic DNA on an N⁺ membrane.
2. Blot the membrane at 80 °C for 30 min.
3. Block non-specific antibody binding sites by soaking the N⁺ membrane in 5% BSA in TBS-T for 1 h. Use a 10 cm Petri dish as a reaction chamber at room temperature.
4. After washing 5 min three times in TBST, incubate the membrane with a mouse anti-5-methylcytosine (5-mC) monoclonal antibody (1:1,000) in TBS-T at 4 °C overnight.
5. Wash the membrane for 5 min three times in TBS-T, and then incubate with a secondary antibody, HRP-conjugated sheep anti-mouse immunoglobulin-G (IgG) (1:5,000) in TBS-T for 1 h at room temperature.
6. Wash the membrane for 5 min three times in TBS-T.
7. Add the enzyme substrate to the membrane and incubate for 5-10 min. Visualize the secondary antibody signal using a chemiluminescence kit according to the manufacturer's instructions.

Materials

Name	Company	Catalog Number	Comments
Reagents
DMEM	Gibco Inc.	11965–092	Warm in 37 °C water bath before use
Phosphate-Buffered Saline(PBS)	HyClone Inc.	SH30256.01B	D-PBS, free of Ca2+/Mg2+
FBS	Gibco Inc.	10099-141
0.25%Trypsin/EDTA	Gibco Inc.	25200-056
1%Penicillin-Streptomycin	Gibco Inc.	15140-122
Chloroform	Mallinckrodt	4440
Isoamyl Alcohol	Sigma	I-3643
Phenol	Gibco BRL	15513-039
Proteinase K	Gibco BRL	24568-2
TAE buffer	Bio Whittaker	16-011V
Distilled Water	Gibco BRL	15230-170
1 M Tris-HCl	Biosharp Inc.	BL514A
Tween20	Biotopped Inc.	C58H114O26
BSA	Proliant Inc.	68700
Collagenase, Type II	Sigma-Aldrich	C6885
Equipment
Cell Strainer (40μm nylon)	FALCON Inc.	352340
Hemocytometer	ISOLAB Inc.	075.03.001
Falcon 100 mm  dish	Corning	353003
Centrifuge Tubes	TPP AG	91050	Gamma-sterilized
High-speed centrifuge	Eppendorf	5804R
ThermoMixer	MIULAB	MTH-100
Carbon dioxide cell incubator	Thermo scientific	3111
Chemi-imaging Analyse System	UVITEC Cambridge	ALLIANCE