2 replies to this topic

[kaika87]

Dear all,

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

[chason]

Fragmentation shouldn't affect yield or quality.  I'd run a sample of your DNA on a gel with a 100bp ladder to check how fragmented it is.  There could also be inhibitors that are inherent in FFPE samples in your DNA that are not affecting the absorbance readings.  Diluting the DNA more and/or using BSA in your PCR reactions might work.  Whether it is fragmentation or inhibitors, using primers that amplify a smaller sequence can help as well.

[Maddie]

Your DNA will be fragmented for sure. I'd simply re design primers to amplify ~200bp.
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.
Good luck.
Maddie