For the optimization of primers for bisulphite sequencing, I extracted DNA from blood which was followed by its bisulphite conversion. Through gradient PCR, all primers were standardized successfully for their optimum annealing temperature. But, when I ran PCR reactions using bisulphite converted DNA from FFPE samples, I am not getting results for most of the primers (when visualized on Agarose gel). Degradation and fragmentation of nucleic acid in FFPE can be a major factor which I tried to rectify by reducing the digestion time and increasing the DNA input in PCR reaction.
Can anybody point out some modifications that can be done in order to achieve desired results from FFPE samples.
@https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211400
Is there a reason you are running bisulphite conversion, and have you looked into using the enzymatic methylation kit by NEB?
This is much more gentle to DNA than the harsh bisulfite treatment?
Hello,
What method are you following to extract DNA from FFPE samples? Our lab has standardized a protocol wherein we have successfully isolated DNA from archived FFPE samples as well as archived H&E slides. You can have a look at our paper.
Hope this helps!
Article DNA extraction from archived hematoxylin and eosin-stained t...
Pushkal Sinduvadi Ramesh for DNA extraction from FFPE, I deparaffinize the cut sections using xylene and decreasing gradient of ethanol followed by extraction using kit from Qiagen (QiAamp FFPE DNA kit). I am currently planning to use mineral oil as a substitute of xylene.
Shreya Srivastava Yes you are doing it right. I too have used the kit from Qiagen but felt Macherey-Nagel kits were better in terms of pricing as well the DNA yield. You can also try modifying the phenol-chloroform isoamyl alcohol method that will save you a lot of money.
About using the mineral oil- I would say refrain from it. I too used it after the literature survey but did not serve the purpose as expected.
Pushkal Sinduvadi Ramesh Can you share the phenol-chloroform isoamyl alcohol method for extraction? And can I directly proceed to phenol-chloroform extraction after deparaffinization step?
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