7 replies to this topic

[strepto]

[COLOR=blue]
Hi,
We are seting up the lab for study on methylation causing transcripsional silencing of tumor suppresor genes.
One of the reason of not getting a PCR product in a methylation specific PCR may be improper bisulphite treatment. Otherwise the DNA may not be  actually methylated. We are using standard with known methylated CpG island, but we are not getting MSP product .
Can anybody help us how to check the bisulphite treatment is correct or not which we strongly doubt?
Is it anyway possible to check without going for sequencing?
Thanks in advance.
PSS

Edited by strepto, 01 March 2005 - 04:13 AM.

[labtechie]

Hi-- Unfortunately I think it is becoming the consensus that MSP is prone to artifact and the bisulfite sequencing is the way to go.  If you are not getting a product with your known methylated CpG island, something must be wrong... PCR conditions, or even that one crucial (for amplification) CpG site is not methylated.  That is the problem with a site-specific method like MSP.  If you are just getting started, you might want to seriously consider switching to the bisulfite treatment then amplification using primers with no CpGs then sequencing-- I spent five months trying to avoid sequencing and it turns out that it's just really the best way to go scientifically.  Look under my "the actual bisulfite treatment" thread for a really good protocol.

Best of luck,
Ellen

[pcrman]

The problem could be in the bisulfite treatment or PCR.  You said you are doing MSP, then you must have USP primers. Did USP work on your DNA? Can you post your PCR condition? Did you use some kit for DNA treatment and How did you purify your DNA after treatment?

[methylnick]

pcrman, on Mar 1 2005, 05:15 PM, said:

How did you purify your DNA after treatment?

Another important thing is, how did you purify your genomic DNA prior to bisulfite treatment, clean, protein free gDNA is very important!!!

Nick

[strepto]

pcrman, on Mar 1 2005, 05:15 PM, said:

The problem could be in the bisulfite treatment or PCR.  You said you are doing MSP, then you must have USP primers. Did USP work on your DNA? Can you post your PCR condition? Did you use some kit for DNA treatment and How did you purify your DNA after treatment?

Hi,
We use Qiagen DNA purification kit fpr genomic DNA purification prior to bisulphite treatment and also after treatment. We don't use any kit for bisulphite treatment.
In fact right now the bisulphite modification is working, however, there is  very low yield of the amplification product and sometime there is no yield.
We are suspecting loss of DNA.
Can you suggest the reasons of not getting the yield.
Thanks.
PSS

[methylnick]

Have you performed a PCR with your USP primer set?

It would help us all if you can also send us your conditions....how many cycles are you performing?

If you are getting a low yield of PCR amplicon, do you have enough for cloning for seqeuncing, direct sequencing or even a restriction digest, because if you seqeunce the product, it would give a clear idea of your conversion efficency, you want all unmethylated C's to be T's. A quick way is to throw a bit of MspI to your amplicon, I assume because you are working with a CpG island, without knowing the exact DNA sequence you are working with the liklihood of it containing a MspI site is high. MspI can test for the conversion of CCGG to TCGG quite easily. If you get a cut that means your bisulfite conversion was not optimal if you don't you know that all CCGG sites within your island (regardless of methylation status) have been converted.

Good luck strpo!

Nick

[pcrman]

Quote

We use Qiagen DNA purification kit fpr genomic DNA purification prior to bisulphite treatment and also after treatment.

I am not sure if Qiagen kits are optimized for the cleaning up of bisulfite modified DNA and we have never use them for such purpose. The most commonly use kit is Promega's Wizard DNA cleanup kit. I think that's the reason you are losing DNA.

Nick, do you think Qiagen kits can be used following the modification?

[methylnick]

pcrman, on Mar 15 2005, 03:47 PM, said:

Nick, do you think Qiagen kits can be used following the modification?

Pcrman,

not too sure. I am not a real fan of anything from Qiagen, their kits in my hands don't work very well (ie i don't get good yields from using them). I am not too sure if the kit is column or resin based, can you answer this for us Strepo?

The wizard is great for desalting the bisulfite reaction it works very well in my hands.

I have many bad things to say about qiagen , but i better not go any further.

Nick  

Edited by methylnick, 15 March 2005 - 07:41 PM.