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| CpG Island Methylation in Aging and Cancer |
DNA methyltransferase Assay
This protocol was written by Jean-Pierre Issa, based on Adams et al.*
Kam-Wing Jair has made some useful shortcuts that work well if you are careful. Here is his version of the assay.
This assay can be used to measure activity in small, snap frozen pieces of tissue (e.g. colon polyps), or in cultured cells.
1. Homogenize cells/tissues
For cells in culture: Harvest log phase growing cells (trypsinization is OK), wash twice in PBS, pellet by centrifugation (2000 rpm, 5 min in table top centrifuge, or 3 min in microfuge), resuspend carefully and completely in Lysis Buffer (see solutions), incubate at RT 5 min with frequent vortexing, and freeze in ETOH/dry ice bath. Repeat freeze/thaw cycle twice (3 times total) and store at -700C until ready to quantitate proteins. Try to suspend 105 cells/l5 ml LB. The number of cells need not be exact, but you need a minimum of 5x105 cells in 75 ml to have accurate results, and if you have too many cells in a small volume, they will not resuspend or lyse well.
For frozen tissues: Homogenize well in LB (200-300 ml is appropriate for colon biopsies and polyps), using a tissue homogenizer if possible, pass the solution through a 20 gauge needle x2, followed by a 25 gauge needle x2, and freeze in an ETOH/dry ice bath. Repeat freeze/thaw cycle x2 (3 times total), and store lysate at -700C until ready to quantitate proteins.
2. Quantitate Protein Concentration
Quantitate proteins in 15 ml aliquots of lysates (in duplicate) by using a Bradford assay (I have been using the Biorad kit), using BSA as standard. Don't forget to add 15 ml LB to each BSA sample and to the blank tube. Use the same LB batch as that used to lyse the cells. 105 cells in 15 ml usually yields 10-25 mg protein. If you need the gory details: Thaw unknowns (one at a time), aliquot 15 ml into two 1.5 ml tubes, add 200 ml Biorad stain (undiluted) and 785 ml 0.15M Nacl and mix. Prepare standard curve by mixing 2.5, 5, 10. 15, 25 and 30 mg BSA with 15 ml LB, 200 ml Biorad stain and 785 ml 0.15M Nacl. Also prepare blank tubes with LB, stain and NaC1 but no protein. Set Spectrophotometer on OD595, zero using blanks, measure OD of standards and unknowns, and read unknown concentration from standard curve. It is convenient to aliquot 30 ml of lysate at the same time you aliquot for the protein assay, and freeze this aliquot, to be diluted later.
3. Dilute samples
After quantitating proteins, dilute 30 ml aliquots by adding LB. For cultured cells, I have been diluting to 0.5 mg protein per 15 ml LB. For frozen tissues, I have been diluting to 5 mg protein per 15ml. This assay is linear up to 4 mg protein from cultured cells and 10 mg protein from whole tissue, but the linearity is lost at lower concentrations if you use a bad batch or an old batch of SAM, and it is therefore better to work with the lowest concentration that gives interpretable results. For the same reason, it is best to have all samples assayed at the same concentration, If in doubt about your samples or the quality of SAM, determine activity at two concentrations or do a concentration vs activity cuve prior to assaying all samples.
4. The assay proper:
- Mix 15 m l lysate with 2 m l poly[d(I-C).d(I-C)1 (Pharmacia, diluted to 0.25 m g/m l using low TE), and 3 m l SAM (Amersham TRK 581). To avoid bubbles, pipet all solutions onto the wall of the microfuge tubes, and spin 2-3s to mix the samples.
- Incubate at 370C for 2 hours.
- Add 350 m l Stop Solution.
- Incubate at 370C for 30 min. (optional).
- Add 350 m l PCI-9, mix by vortexing, spin in microfuge 30s, transfer the supernatant to a new tube and repeat xl (2 PCI extractions total).
- Add 350 m l chloroform, mix by vortexing, spin 30s and transfer supernatant to a clean tube.
- Add 700 m l absolute ETOH (kept at -200C), mix by inverting tubes 2-3 times, and place in -700C freezer for 15 min or -200C freezer for 30 min, The assay can be interrupted at this point, and the samples kept at -200C until ready to proceed.
- Vortex 5-10s
- Spin in cold room microfuge for 15 min.
- Pour off supernatant.
- Add 500 m l 70% ETOH. Vortex.
- Spin in cold room microfuge 10 min.
- Pour off supernatant.
- Pipet off residual ETOH. Alternatively, dry in speedvac for 10 min, but the pellet does not have to be dry to proceed.
- Resuspend the pellet in 30 m l 0.3M NaOH, vortex 5s, spin briefly to bring solution to bottom.
- Incubate at 370C for 45-60 min.
- Spin briefly.
- Spot solution onto GF/C Whatman filter papers (2.4 cm2 fits best the current manifold).
- Dry 5 min. in 800C oven.
- Place filters on manifold with suction off.
- Pour 1-2 ml ice cold 5% TCA with BSA (3 ml of 10 mg/ml solution for 500 ml TCA).
- Wait 10 sec.
- Turn on vacuum. Wash filter with 2-5 ml TCA/BSA, followed by 2-3 ml 70% ETOH.
- Dry in 800C oven for 5 min.
- Place filters in scintillation vials.
- Add 5 ml Econofluor.
- Shake manually 5s.
- Count in liquid scintillation counter (3H channel)
- Et voila !
5. Troubleshooting:
Any deviation from this protocol will be sternly punished ! But seriously, do not assume that some of these steps are superfluous; I have been there ! e.g. Two PCI extractions really are necessary, drying of filters in oven is essential etc. If you think you can shorten/improve this assay, please do, but make sure you check your modification against this assay, checking both positive and negative controls. Run all samples in duplicate. Because the quality of SAM varies tremendously from batch to batch, run a positive control with each assay. If we receive a new SAM batch, try to compare old vs new batch in the same assay, using the same sample (this would greatly simplify correction factors). Also, if you are the first to use a new SAM batch aliquot it into 75 ml aliquots because repeated freeze/thawing of SAM accelerates its degradation. Finally, run a negative control with each assay. This should be the same sample as the positive control, with SAM, but with no dIdC. If your negative controls are too hot: Did you include dIdC by mistake ? Is the grid of the manifold contaminated ? (you can check by using a blank filter, washing with TCA, drying and counting in Econofluor) Also, a longer NaOH incubation might reduce this problem some. But remember, the higher the activity, the higher the negative control will be because of methylation of endogenous DNA. By the same token, a higher cellular protein concentration will also lead to a higher negative control for similar reasons (+ protein methylation). If your positive controls are too low: Did you deviate from protocol ? (e.g. not drying will definitely lower counts). Did you lose pellet during 70% ETOH wash (yes it happens to the best of us - and the worst). Are you dealing with a bad or degraded batch of SAM ? (so far, one batch was bad because it stayed in the freezer for 6 months before being used, one batch was worthless - it was replaced free of charge by Amersham, 2 batches were average and one batch was great). If none of the above, may be the gods were against you on that day-repeat ! One last thing: (promise - I am getting tired of typing). I usually run 24 samples at a time (ie 1 negative, 1 positive controls, and 11 unknowns in duplicate). 24 is because the microfuge will not take more samples, and doing more than that on a single day is - I think - crazy. It takes 30 min to set up reactions, 2 hours incubation, 1-2 hours stop/PCI, and 2-3 hours to finish the assay.
GOOD LUCK.
6. Solutions
LYSIS BUFFER
Stock: 72 8 ml H2O
5 ml 1M Tris pH 8.0
200m l 0.5M EDTA
1 ml 1% Na Azide
10 ml Glycerol
1 ml Tween 80
(Store at RT)
Add Fresh: 5 m l 0.2M DTT per ml LB, 5 m l PMSF per ml LB (PMSF stock: 12 mg/ml in Isopropranolol)
STOP SOLUTION
Stock: 50 ml H2O
10 ml 10% SDS
400 m l 0.5M EDTA
4 gm 4-Aminosalicylate (Na salt, Sigma)
5 ml Butanol
12.5 ml 1M NaCl
8.3 ml Salmon testis DNA (sonicated, stock: 3 mg/ml)
H2O to 100 ml
(Store at 4o C)
Add Fresh: Proteinase K, 50 m l of 20 mg/ml stock per ml stop solution
*Adapted from: RLP Adams et al, Journal of Biochemical and Biophysical Methods, 22:19-22, 1991.