real-time PCR for quantitation of MSP - (Jun/13/2006 )
I got the bands using both methylation-specific and unmethylation-specific primers. I doubt that maybe the CG is not totally transferred to TG after bisulfite treatment, is it one possibility?
Another possibility is the template DNA is a mixed DNA from different types of cells. Actually, the tissue includes 4 types of cells. My boss suggests that I should use real-time PCR to quantify the methylated and unmethylated template. But I am wondering the housekeeping gene normally is unmethylated. Should I compare the amount of M and UM PCR products only using unmethylated housekeeping gene as control in realtime PCR? Is it feasible?
Hope to get your help. Any suggestion is highly appreciated.
I think it is a good idea to use a reference gene (not necessarily a housekeeping gene) as a control for starting DNA amount.
You may want to read the following two papers to find more information.
Huh, very old topic, but I have one question related to it:
If I use a nested PCR strategy for MSP - meaning a common outer primer for the first round and then specific M and U primers for the second round, than I won't really need a reference gene (because I assume that I have the same starting amount of PCR product in both reactions) - am I right? If the efficiency of PCR is equal for U and M, than I should be able to calculate the amount of methylation according to this formula:
%methylation = (1-[2CtM/(2CtM+2CtU])*100
Do you agree? Or am I missing something?
It should even be possible to adopt the Paffl correction this way:
EM= Efficiency of M primer
EU= Efficiency of U primer
%methylation = (1-[EMCtM/(EMCtM+EUCtU])*100
What do you think?