DNA extraction from FFPE?
Posted 12 November 2009 - 01:18 AM
I'm not sure whether this has come up before and I'm sorry if I may sound 'noob' with this question.
But I'm doing DNA extraction from archival FFPE tissues which spans from 2-5 years old. I've done the extraction and carried out a PCR run with beta actin housekeeping primers for a 500+ bp PCR product but failed to observe any bands.
I read that its possible that the FFPE tissue may have been fragmented to sizes <500bp in size. So to verify DNA presence I used Nanodrop to check the yield and the quality. Turns out that there's a concentration of around 100ng/ul with 260/280 of 1.97 and 260/230 of 1.73 which to me seems fine.
My question would be, even after carrying out PCR using the housekeeping primers, I couldn't see a band whereas the positive control I used (which was DNA from swabs BTW) shown a band at the 500+ region. Could there be any other reason asides from fragmentation? And if that's the only explanation, what could I do to verify that the DNA has indeed been fragmented??
And the follow up question will be, should I design another primers which may detect smaller bp sizes in order to help detection of the fragmented (if so) DNA?
Thanks in advance
Posted 12 November 2009 - 12:26 PM
Posted 17 November 2009 - 04:24 PM
You can also check that there is no inhibitor left in your extract by mixing your extract and fresh DNA.
BSA is good. Just make sure it's a non-acetylated one 9so you can use a lot).
if it still doesn't work, I'd add a few PCR cycles and maybe a bit more Taq.