Nested MSP - (Dec/19/2005 )
So I'm playing around with a nested MSP and was curious how many cycles of amplfication are typically used in each round? I tried 30 + 30 but when the products were run out on a gel I seemed to have faint M band in my Unmethylated control and a faint UM band in my methylated control.
What would be the ideal cycle conditions to try to eliminate the background from unspecific priming? Would I be better off adjusting cycle number? or the annealing temp (from papers)?
ideally you would want to run it on a q-PCR setup with SYBR Green.
but lowering your cycle number to 20-25 will rid you of the faint band.
The reaction that I mentioned earlier was done using the Roche Lightcycler 2.0 with Sybr green in my master mix. Interestingly the crossing point on all my samples was > 30 on methylated primer with unmethylated control and vice versa on my methylated control so it seems as though quantitative data is possible. But when I run out my products on a gel I had those faint background bands that I mentioned earlier.
Is there any way of getting actual quantitative data from running SYBR green q-RT PCR on msp primers? How would you go about normalizing the data, to account for slightly different amounts of DNA after bisulfite treatment if you lack a accurate means of quantifying the small amount of resulting converted DNA?
Well as quantitative as you can get it, you would start off with the same amount of DNA for bisulfite conversion and you would assume that you would yield equal amounts after conversion for qPCR.
you will need to perform a (serial dilution ) titration curve to determine the quantitative amounts in your experiemnts, of course this will be difficult if you don't have a reference to start with. Furthermore, things are complicated when you have in your sample, a mixed population whereby there will be some DNA molecules that are unmethylated in an otherwise methylated population (hence the faint band you are seeing) the faint band that you see will have a higher Ct value than your stronger band.
It make things clearer if you can post a photo of your gel.