real-time PCR for quantitation of MSP - (Jun/13/2006 )

Hi, everyone:

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.

Linksky

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.
http://www.ncbi.nlm.nih.gov/entrez/query.f...l=pubmed_docsum
http://www.ncbi.nlm.nih.gov/entrez/query.f...l=pubmed_docsum

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-[2^CtM/(2^CtM+2^CtU])*100

Do you agree? Or am I missing something?

It should even be possible to adopt the Paffl correction this way:
E_M= Efficiency of M primer
E_U= Efficiency of U primer

Formula:

%methylation = (1-[E_M^CtM/(E_M^CtM+E_U^CtU])*100

What do you think?

Krümel