FFPE archival materials and MS-PCR - (Oct/15/2007 )

DNA extraction from FFPE archival materials usually gives degraded DNA, so let to discuss:
1)How to improve extraction method or quality of extracted DNA?
2)Will the results of methylation specific PCR be influenced by such degradation?
3)Which kind of positive controls can be included to be assuring that the template is OK for MS-PCR?

hi siamak,

FFPE is what I assume to be formalin fixed parafin embedded?

Haven't worked with such samples but there are a smattering of papers that have used it in the literature.

I would say because the DNA is so degraded the MS-PCR assays should be designed such that they amplify a very small region of sequence so there is a higher chance of picking it up (100-250bp).

As for positive controls, this would depend on your tissue type. You can select for genes of known methylation status in your tissue and test those in your assay.

good luck

nick

siamak,

for positive breast tissue controls, I think the best place is to start with breast cancer papers looking at methylation for candidate genes that could be used as controls. For such DNA I would reccomend Genetic Signatures Methyleasy Xceed kit as a cheap way of converting your DNA.

Nick

imprinted regions?