10 replies to this topic

[InduS]

Hi guys,
I need some help regarding MSP?
-Is methyl specific PCR metod genuine enough to detect the presence of Methylation? somtimes during normal PCR the DNA template don't amplify due to various reasons. If this happens in MSP can this produce false results?
-Is Bi-suphite sequencing better is this regard?
-About the specificity of primers....how do I know the primers have bound to the promoter regions (I've heard that promoter regions are highly similar and hence the primers have to be carefully designed.?)
-ITs the first time ill be doing is MSP. Please tell me the experimental layout for this procedure. ie- how many controls for methylated and unmethylated samples should be taken?
-Regarding primers... do I need to order two sets? One before bisulphite modification and one after the modification? Do I need to do this simultaneously for comparison?
My questions may sound weird but I've done this before and I need to write cost estimation for this procedure

Thanks in advance

[pcrman]

-Is methyl specific PCR metod genuine enough to detect the presence of Methylation? somtimes during normal PCR the DNA template don't amplify due to various reasons. If this happens in MSP can this produce false results?

Certainly this concern is real. In my opinion MSP is very error prone.

-Is Bi-suphite sequencing better is this regard?

Yes, it provides more accurate methylation mapping with higher resolution.

-About the specificity of primers....how do I know the primers have bound to the promoter regions (I've heard that promoter regions are highly similar and hence the primers have to be carefully designed.?)

No, your statement that promoter regions are highly similar is not true. What is true is that after bisulfite modification, DNA complexity is reduced and primers should be carefully picked to avoid ambiguous amplification.

-ITs the first time ill be doing is MSP. Please tell me the experimental layout for this procedure. ie- how many controls for methylated and unmethylated samples should be taken?

Ideally, you should have full methylated and unmethylated DNA as controls.

-Regarding primers... do I need to order two sets? One before bisulphite modification and one after the modification? Do I need to do this simultaneously for comparison?

You should have two sets of primers: one set for methylated DNA and the other for unmethylated. The two sets of primers should be designed in a way that they preferentially amplify modified DNA besides their specificity for either methylated or unmethylated DNA. You don't need primers that amplify unmodified DNA.

[InduS]

Thanks for the reply. Due to lack of funds i'd have to stick with MSP. Number of studies have been conducted successfully by MSP, I'd have to stick to that. Please tell me how I can optimize my results.

[pcrman]

If you want to stick to MSP, you really need to optimize your PCR conditions to make sure any amplification is specific. If you have a problem obtaining PCR product, you can first use BSP or universal primers to amplify the modified DNA, and reamplify using MSP primers.

[genie]

Thanks for the reply. Due to lack of funds i'd have to stick with MSP. Number of studies have been conducted successfully by MSP, I'd have to stick to that. Please tell me how I can optimize my results.

try using the MethPrimer software for both MSP and BSP primer designing.

[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..

[methylnick]

do you have a mixed population of cells in your sample? Normal and Tumour?

[katrina]

actually there is no micro dissection system in our institute and we take the tissue purely based on the pathologists confirmation. i am also following the exact protocol for modification of DNA as well.. i really don know wht is the problem. the tissue specimen might be heterogeneous but don know if all the genes would give the same pattern.

thank you for the reply.

[katjaze]

Hello guys!

I am also brand new with the MSP problematic..Currently I am stucked with the standardization of MSP/USP for genes of interest (even I obtained primer sequences for both MSP and USP from the published articles and PCR conditions for both nested PCR and MSP/USP)..As Katrina, I usually get the bands both methylated and unmethylated for the same sample ?! My advisor told me that this is usually happen when PCR conditions are not adequatly optimized...We also obtain tumour tissue from the pathologists who did the tumour dissection. Beside that, I know that it would sound very silly, but I have to optimize the PCR conditions without having the full methylated and unmethylated control!!! Is that possible to achieve??? If yes, how and could I obtain convinced results doing that?

Waiting for the replay!
Best, Katja

Edited by katjaze, 20 April 2012 - 02:08 PM.

[amrwaly]

I have read many papers where methylated and unmethylated control were not used... DNA was bisulfite converted and they designed two set of primers for methylated and unmethylated sequences and used SYBR- Green master mix... in other words the did Quantitative Real-time MSP... Ct values where obtained and percentage of methylation was calculated for each tissue by a mathematical formula... For more details check out the following CANCER RESEARCH paper: Hypermethylation of let-7a-3 in Epithelial Ovarian Cancer Is Associated with Low Insulin-like Growth Factor-II Expression and Favorable Prognosis...
Check the Materials and methods section: (Analysis of DNA methylation)

[katjaze]

Thank you Armwaly for your reply and the article! That is also a good idea for MSP, but I have to do PCR, not Real Time, unfortunatelly..Anyway it is still good to hear that my job is possible to do, even without positive/negative methylated control!

Best, Katja