22 replies to this topic

[spjgjsm]

Hi Ellen

I just did 2 TE washes and 1 water wash and it seemed to be fine.

I have some universally methylated control DNA and this showed methylation using the protocol, so it is fine (although like you I found no methylation in my actual test samples !)

I haven't done much sequencing on the samples yet, so can't comment on the non-converted C's, but will let you know as soon as I get round to it.

I also didn't enzyme digest and don't appear to have problems.

Cheers

Jon

[labtechie]

Thanks everyone for all the help.  
Nick: when you say "spike" do you mean that I should have the plasmid in the same bead as my sample so as to ensure that it is getting the EXACT same treatment?  Or can I have it as a different bead and just do the same protocol at the same time?
Jon (and anyone else): When you get no methylation in your test samples, I assume these are CpG islands yes?  I figure mine is a perfect negative result, but my boss is not happy.  He thinks that there should be some methylation somewhere even though I show him the literature that most CpG islands are NOT methylated at all.  Anyone have any really good literature on this they can point me to?
Ellen

[methylnick]

labtechie, on Feb 24 2005, 11:24 AM, said:

Nick: when you say "spike" do you mean that I should have the plasmid in the same bead as my sample so as to ensure that it is getting the EXACT same treatment?  Or can I have it as a different bead and just do the same protocol at the same time?


He thinks that there should be some methylation somewhere even though I show him the literature that most CpG islands are NOT methylated at all.  Anyone have any really good literature on this they can point me to?

Ellen,

yes, ideally you should have the plasmid in the same bead as your sample to ensure you are getting the exact treatment for both DNA's.

As for your island of interest, is it part of a promoter of a gene? if so, has the gene been switched off? There are cases in the literature where the CpG island promoter is unmethylated and yet switched off, there are also cases where the island is methylated and the gene is expressed. I have found such a case!!  

in terms of literature, there are a number of good review papers published by Bird's Group and Feinberg's Group on the topic. Can't tell you off the top of my head now though sorry!  

You are right to tell your supervisor that most CpG islands are not methylated. You may have to look at the chromatin environment surrounding your gene as chromatin proteins can modulate gene expression, that maybe a whole new kettle of fish!

Good luck with it all!

Nick

[spjgjsm]

Ellen

I think your results are probably fine - if the CpG's you were looking at were methylated then they wouldn't be converted.

It is possible there could be very low-level methylation - perhaps test this using a quantitative method like MS-SNuPe - if the level was very low (e.g. 5%) you may not pick a methylated clone from the number you are sequencing.

On a technical note about the bead protocol - how long do you think the treated DNA is stable for after the protocol (if stored in a fridge in TE)??

cHEERS

jON

[labtechie]

Jon-- I've used treated DNA beads up to about three weeks after and they were fine-- some protocols I've read say five days.  I don't have the technical expertise to give you any better advice than that though.

Thanks, methylation buddies, for backing me up on the "CpG islands are not methylated" debate with my boss.  I'll have to do a positive control.  But if I do my own methylation of a plasmid, how do I know it worked?  I mean, if I methylate the plasmid, spike my samples, then treat, then sequence the plasmid and the CpGs are not methylated, how do I know if the methylation step failed or if the treatment is too strong?

So complicated.... thanks for all the help,
Ellen

[methylnick]

labtechie, on Feb 28 2005, 08:58 AM, said:

But if I do my own methylation of a plasmid, how do I know it worked?  I mean, if I methylate the plasmid, spike my samples, then treat, then sequence the plasmid and the CpGs are not methylated, how do I know if the methylation step failed or if the treatment is too strong?

Hey there Ellen,

All is not lost, if there is a will there will always be a way   .

Okay so you should choose a plasmid that contains HpaII/MspI sites (ie: CCGG). To check if your SssI methylase experiment worked, digest the treated plasmid with HpaII. HpaII is methylation sensitive and therefore you will not get digestion. You can also digest the same plasmid with MspI which is methylation insensitive and you would expect full digestion of all sites within the plasmid. Of course if you perform the digests with untreated plasmid (isolated from DAM- bacteria) then it will digest with both HpaII and MspI. (Of course you can use other methylation sensitive isoschizimers if you choose).

You do not have to perform sequencing of your PCR amplicon post bisulfite treatment. A neat way of checking if your positive control has been fully converted is to digest your amplicon with restriction enzymes. Again like above, if you were to digest your amplicon with MspI you will get no digestion if your bisulfite conversion has worked because all CCGG sites will be converted to either TCGA (if methylated) or TTAA (unmethylated), So that is enough to say that your bisulifte has worked efficently, To check if your methylase worked assuming that it did, all CCGG sites would be converted to TCGA after bisulfite and PCR amplification. So if you were to digest with TaqI, these sites will be cleaved. (This is of course cheaper than to directly sequence).

Hope this helps you, I am sure your supervisor will be impressed with the way you are attacking the problem!

Nick

[Jackey]

Could I ask a question concerning the use of SssI? We seem to be getting very low efficiency when attempting to methylate human genomic DNA with this enzyme. It seems to work better on DNA from the cell line MiaPaCa2, which appears to have a high level of methylation of various tumour suppressor genes by itself, than on lymphocyte DNA, but still a strong band for the unmethylated reaction remains in the PCR. I used two units of SssI for 1 microgram of DNA, and did the reaction for 2,5 hours at 37°C with the buffer conditions recommended by the manufacturer. Should I increase the amount of enzyme, leave the reaction longer or add more SAM?

Thanks for any advice.

[katrina]

i am a Phd student.. i have been working on hypermethylation of 12 different genes (6 DNA repair genes and 6 TSGs) in ovarian carcinoma. i have standardised 5 genes out of 12. i have used the nested pcr method for all the genes.. have selected the primers for nested, MSP and USP from published articles. and also the conditions for the preparation of master mix from the articles. i have used normal taq DNA polymerase. i have got the desired amplicon size after the pcr when observed in 3% agarose gels.. the amplicons correspond to the primer product of methylated and unmethylated primers. have standardised the protocol using 2 carcinoma samples, 2 benign samples and one positively methylated control and one negative water control. so far things were working fine..

my problem now is out of the 5 genes which i have a standardised, i have got bands in both methylated and unmethylated samples in all the samples except negative control. even in the positive control prepared by treating with SssI methyltransferase both methylated and unmethylated bands are seen. so i don know what is the problem.. why am i getting bands in both for all teh genes.

is my bisulfite modification improper?
is my PCR not working??? this might be a rare possibility because i am getting desired product and also have followed the protocol according to published articles...

i don have a microdissection system in my institute so wont be able to isolate tumor tissue separately.. so the tissue is heterogenous.. this may be the reason for seeing the bands in both. but why is seen in all the genes???

please help me with this... i need to proceed with the protocol with a larger sample size. if i get similar results in all then the data wouldn make sense.. kindly help me as it would help me in carrying out my research work at ease..

waiting for the reply as soon as possible...

katrina..