start a methylation study - (Jul/06/2006 )

Hello,

I'm a little lost and am running out of time. Have 2 genes which are silenced during transcription and i have to postulate a reason for this phenomenon. I know that methylation is a possibility...this is what i have done this far:

1) using Methyleasy from Human Genetic Signatures for the DNA bisulphite Modification...is this good? Anybody else tried it before?

2) I tried 5-aza-CdT treatment with 2 liver cell lines but didn't get any result as even by positive control didn't show any difference in expression. Anybody can suggest any reason for this? I added 2M and 5M concentration and dissolved it in DMSO.

3) I also used MethPrimer to design MSP primers but don't seem to notice any real pattern for one of the genes and the other has no band just a smear...i am already using a double pcr and have varied the annealing temp from 50 to 59 degrees without much success...

Have used MethylPrimer express to design some new primers and will be doing the treatment again today...any help would be greatly appreciated. Thanks.

cheers,
Gai

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Hi Gai,

The learning curve for methylation study is usually long so be patient and finally you will get it to work.

>>1) using Methyleasy from Human Genetic Signatures for the DNA bisulphite Modification...is this good? Anybody else tried it before?

Most DNA mod kits on the market should work. Personally I have only used a kit from Chemicon.

>>2) I tried 5-aza-CdT treatment with 2 liver cell lines but didn't get any result as even by positive control didn't show any difference in expression. Anybody can suggest any reason for this? I added 2M and 5M concentration and dissolved it in DMSO.

The concentrations you used seem to high to me. We use concentrations ranging from 1-10 uM. 5azaC can be also dissolved in PBS or medium. Some cells require longer treatment such as 1-2 weeks. Sometimes you have to treat cells non-consecutively (i.e., treat cell every other day). If no expression is induced after trying different treatment protocols, probably methylation is not responsible for the silencing. Does your gene has CpG island at its promoter? Absence of CpG island may also indicate that methylation is not involved.

>>3) I also used MethPrimer to design MSP primers but don't seem to notice any real pattern for one of the genes and the other has no band just a smear...i am already using a double pcr and have varied the annealing temp from 50 to 59 degrees without much success...

PCR on bisulfite modified DNA is very tricky. First make sure your template DNA is good. You can try your DNA using some primers known to work. Hot-start your PCR is necessary and using some hotstart taq such JumpStart Red Taq from Sigma helps a lot.

Good luck.

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Thanks so much pcrman for your prompt reply!
I realised i had typed the wrong drug dosage. I had used 5µM and 10µM concentration on my cells. Do you think dissolving it in DMSO was alright? I dissolved the drug in DMSO and am storing it at 1mM concentration. Will it degrade in anyway?

One of my genes has a CpG island about 1500bp away from the start codon and the other is just before the 1st exon. Methylation can be a possibility right?

Thanks again for your help!!!

gai

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Yes you can dissolve 5azaC in DMSO and store at -70C but avoid multiple freeze-thaw cycles.

So your genes are CpG island containing genes and are likely affected by methylation.

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Thanks again for your advice...now i feel like i'm finally getting somewhere with my research.

Just a quick qn: I am currently using a gene for positive control that worked on my methylated products. A band only appeared with the methylated primers and not with the unmethylated primers. To confirm the results, i used the same set of primers with my untreated genomic DNA and ran a PCR with the same programme.

My results were confusing as i had bands with me unmethylated primers and not my methylated primers...isn't that wrong?

Gai

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Either M pair or U pair should not amplify anything from untreated DNA. If contamination can be excluded, then inadequate specificity for the U primers is the problem. Try raise the annealing temperature a little bit to see if you can get rid of the band from untreated DNA.

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I think i might have gotten the concept wrong but from what i thought, there should be a band with the methylated primers on untreated DNA as well as the sequnce is the same for the methylated region in treated and untreated DNA right?

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I managed to get the 5-azaC treatment to work but when i re-did my bisultfite treatment to confirm my MSP results, it didn't work. Ended up gettiing smears with either the methylated primers or the unmethylated primers. Is that normal? Does it mean my DNA is dirty?

When i tried my BSP primers i had the same problem with one of the genes..really hope you can help me solve this problem..thanks

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hi gai, i agree with pcrman, you should NOT be getting a band in your untreated DNA,

you M primer sets may amplify untreated DNA if your Tm in you primer is not optimal, smears would indicate a Tm that is too low, I would suggest increasing the Tm like pcrman has said, conversly if you haven't seen anything in your other PCRs that have not worked, this could be indicative of a Tm that is too high and I would suggest lowering them, good luck!

nick

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Hello again...i'm very confused. I truly hope you can help me clear this. The last time i ran a a MSP pcr for my gene i got the result i wanted with a band w the methlated primers and a smear with the unmethylated primers. I also got a band with my BSP.

Now however, I had to make new samples as the old one started to give me smears. My new samples give smears for both sets of primers MSP and BSP with the same conditions. Could it possibly be my DNA? But my positive control gene still gave consistent results.

I'm so lost..don't know how to proceed. Really hope you can help.

gai

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