Monday, March 8, 2010

Extracting DNA or RNA from FFPE Tissues

BioCompare recently published an overview of the challenges associated with extracting DNA or RNA from formalin-fixed, paraffin-embedded (FFPE) tissues. The article looks at various commercially available options for extracting nucleic acids from FFPE samples, including Epicentre’s QuickExtract™ FFPE DNA and RNA kits, and the MasterPure™ Kits. The article cites a review of the QuickExtract FFPE DNA Extraction Kit published on BioCompare by Dr. Michael Campa, Duke University, in which he states:
I was attracted to this kit because the protocol required no deparaffinization of the specimen prior to DNA extraction. Deparaffinization is usually accomplished with 2 to 3 incubations in xylene and can be a nuisance, particularly if you have more than just a few specimens to deal with.
Dr. Campa found that all 40 samples he processed using the kit gave him strong bands in PCR, and the gel-purified amplicons provided excellent sequencing results.

RNA can prove even more challenging to extract from FFPE samples than DNA, due to the greater extent of degradation over time. Glenn et al.* compared the performance of RNA isolated from FFPE tissue slices in TaqMan® qPCR assays. They concluded that:
When using FFPE tissue, the MasterPure kit produced the highest RNA yield with no contaminating genomic DNA. RNA yield can be further enhanced by including an overnight Proteinase K digestion. Gene-specific primers enhance reverse transcription and increase the sensitivity of qPCR.
* Glenn, S., Head, K., Teh, B., Gross, K., & Kim, H. (2009). Maximizing RNA Yield from Archival Renal Tumors and Optimizing Gene Expression Analysis Journal of Biomolecular Screening, 15 (1), 80-85 DOI: 10.1177/1087057109355059

No comments: