We have been trying to extract DNA from FFPE (Formalin Fixed Paraffin Embedded) tissues for tumor testing using NGS, but the DNA yield is insufficient to proceed further. Can anyone please share an optimized protocol for FFPE tissue extraction?
It is not clear which is the intent of the experiments. If it is aimed at amplicon resequencing most likely it is not a problem of DNA yield but a problem in the protocol preparation. My strong advice based on our experience is to design a primer pool for the gene of interest and preamplify your sample.
Here it is a useful approach http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963916/
Hi Sujani.
In our lab we use a kit for extraction and purification of DNA called "Magnesil Genomic Fixed Tissue System, from Promega. We obtained excellent results in conventional PCR. Hope that helps.
Hi Sujani,
There are ways to PCR-amplify your DNA prior to some NGS applicaitons, but of course it would be best to improve the yield of the DNA prep. I (and I think most other people) will need more information before I can help:
1. How much FFPE do you have (per sample)?
2. Which NGS applicaiton will you be using? [i.e. Whole Genome? Capture-based (ExomeSeq, Cancer panel(s))? Amplicon sequencing?]
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