RUNX3 Methylation specific PCR not working-please help - (Jun/15/2012 )
I'm just starting out using MSP as part of my MD thesis. I'm trying to characterise methylation status of some of the CpG islands in promoters associated with colorectal cancer.
I've chosen to look at RUNX3 as a start but I'm having real problems with my MSP. I'm using the sigma imprint bisulphite conversion kit and am inputting around 200ng DNA in total per conversion. I'm using some primers I've found used in several papers and have used the exact same PCR conditions but am seeing no bands on my electrophoresis for either the methylated or unmethylated primers. I've tried changing the length of time for the bisulphite conversion- the kit instructions state 90mins, but as that didnt work, I've tried 3h, 6h, 8h and 16h with no success.
I've also tried running the primers at lower annealing temps than used in the papers- they used around 65C and I've tried with as low as 53C without success.
Has anyone got any good advice to help me please? It's getting quite depressing!!!
I would verify that your input DNA was good. You could do this by PCR of the region you're working with before bisulfite modification. Another issue could be using too much template in your PCR reaction. Bisulfite conversion leaves lots of junk, some of which may be PCR inhibitory. Try dilutions of your converted DNA.
HI thanks so much for getting back to me so quickly.
I designed some standard pcr primers to straddle the runx3 promoter and they amplify the region well and i have a strong band on gel electrophoresis. its just the post modification msp wont work.
As part of the conversion kit there is a post modification clean up step which uses a column and several ethanol washes. I'm eluting the dna back into an elution solution provided but im also eluting the column with h20 separately after the first elution to try and maximise yield.
what sort of dna dilutions would you recommend? i'm getting around 20ng/ul of dna from the conversion kit after cleanup.
Also, this may sound like a stupid question, but i'm running the msp annealing stage at 57C to maximise my chance of success but all the papers have used higher temps- around 65C. is it possible that the pcr would actually be more likely to work at these higher temps?
I would try 10x, 100x and 1000x dilutions of your DNA. What enzyme are you using? I'd recommend Taq or a Taq mixture. Other enzymes are better for fidelity, but Taq nearly always works. What is your GC content? You are reducing your GC content significantly (halving it). This can lead to problems with extension at high extension temperatures. Try running the extension at 64C instead of 72C, and lengthening the extention time.
thanks, I'm using Taq. I'm not sure the exact GC- I don't have my book on me now but I guess its fairly high as its full of CpG sites.
I'll give those dilutions and extension times a go and let you know how it goes!
Thanks again for your help- noone else in my lab has done any msp so I'm really grateful for your advice.
tried running some DNA dilutions with a lower extension temp and longer time as suggested- I've got some very faint bands on the x10 dilution. I've also tried running a temp gradient but not much difference. Are there any other factors I could optimise to get this to work?
I had the same problem and I change the enzyme. I use Zymo Taq mix, 2 µL of undilute DNA. Check if the Zymo Research can send you a trial of the enzyme.
If you have a weak band, that is very good news. I'd recommend that you design nested primers and re-pcr the samples showing weak bands.
I changed the taq from standard go taq and used hot start instead. It has worked really well and getting good strong bands where expected. Thanks again for the advice,