Posted 29 March 2011 - 04:33 AM
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
Posted 29 March 2011 - 07:18 PM
Posted 29 March 2011 - 07:55 PM
Posted 30 March 2011 - 05:18 PM
Posted 18 July 2011 - 12:28 AM
try using the MethPrimer software for both MSP and BSP primer designing.
Posted 28 October 2011 - 08:09 AM
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...
Posted 04 November 2011 - 03:29 AM
Posted 05 November 2011 - 09:26 AM
thank you for the reply.
Posted 20 April 2012 - 02:02 PM
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!
Edited by katjaze, 20 April 2012 - 02:08 PM.
Posted 21 May 2012 - 01:20 PM
Check the Materials and methods section: (Analysis of DNA methylation)
Posted 10 June 2012 - 05:09 AM