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DNA Extraction

Posted by J-Baker on 04 Sep 2014 at 00:48 GMT

I'm wondering if you have tried to obtain DNA from tissue samples for rDNA sequencing or NGS? Formalin-fixed and dehydrated tissue should be suitable for commercially available FFPE DNA extraction kits.

No competing interests declared.

RE: DNA Extraction

jamsmitten replied to J-Baker on 04 Sep 2014 at 21:35 GMT

I've successfully extracted DNA from paraffin impregnated formalin fixed biopsies where cross linkage issues can be overcome. Though would the long term storage in 80% alcohol in this case not have allowed too much formic acid to form in the tissue samples for any success with PCR?

No competing interests declared.

RE: DNA Extraction

J-Baker replied to jamsmitten on 04 Sep 2014 at 23:54 GMT

Hi James,

If the fixation was done correctly, the samples would have been transferred from buffered formol to 80% ethanol and then the 80% ethanol would have been replaced at least once (to remove excess water). Therefore, practically all free formaldehyde would have been removed. With a bulk sample like this (large liquid volume, small air volume) formic acid shouldn't be such a big problem. There will also be calcium carbonate from shells acting as buffer against acidity.

I think the authors should try extractions, using other material from the same bulk samples until the protocol works.

Another option would be "Direct PCR". When I have very little starting material (< 100 cells) I don't risk loosing DNA through the extraction procedure. I PCR directly from disrupted tissue using a robust HiFi enzyme (KOD or Phusion) and a large reaction volume (8x25 ul per sample). Again, there are kits for "Direct PCR" from FFPE samples.

No competing interests declared.