Archival formalin-fixed paraffin-embedded (FFPE) specimens represent a valuable resource of clinical specimens that are suitable for a variety of research protocols. Recent advances in DNA and RNA extraction and analysis techniques have extended the utility of FFPE specimens far beyond that of immunohistochemistry. I recently needed to determine the base orientation of a specific genetic polymorphism in 40 FFPE sections and decided to try the QuickExtract FFPE DNA Extraction Kit from Epicentre Biotechnologies. 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.
One interesting aspect of the kit is that it consists of just one reagent, the FFPE DNA Extraction Solution. The extraction procedure consists of incubating the tissue section in 100 µl extraction solution for 1 h at 56°C, then for 2 min at 98°C. The genomic DNA in the resulting mixture is ready for PCR without further purification, or it can be stored at -20°C or -80°C. The extraction protocol can be used on FFPE sections that have been cut and placed into microcentrifuge tubes or affixed to slides. Since the protocol recommends trimming off excess paraffin, the slide method is simpler in that the parts of the section that actually contain tissue are easier to recognize. I used a no. 11 scalpel, which has a straight blade, to scrape approximately 1 cm2 of tissue into one or more curls. I then used the scalpel to transport the curls to a PCR tube containing the extraction solution and scraped the curls into the tube using a clean pipet tip. Since the incubations at 56°C and 98°C are easiest to carry out in a thermocycler, it is convenient to transfer the tissue scrapings directly into PCR tubes containing the extraction solution.
I first tested the extracted DNA in PCR reactions designed to amplify a 300 basepair region of an intron. I used 0.25 µM of each primer and the following cycling conditions: 94°C for 3 min; 40 cycles of 94°C for 15 s, 55°C for 30 sec, and 72°C for 30 sec; 72°C for 5 min. While an agarose gel revealed the PCR to have worked in most instances, it was clear that using too much of the extracted DNA solution wasn’t good. For a 25 µl PCR reaction, 0.1 to 0.2 µl of the DNA solution worked great. Since the volume of extracted DNA per 1 cm2 section was 100 µl, I only needed 1/1000th to 1/500th of the amount obtained to get a strong band on a gel after loading only 2 µl of the reaction. All 40 of my extracted DNA samples have given strong bands following PCR.
Since the PCR reactions gave me a single band on a gel, I purified the amplicon directly from the reaction mix and sent 30 ng each from the first 8 reactions for sequencing. All samples sequenced all the way through the amplicon. The chromatograms contained very distinct peaks with minimal baseline fluctuations. Not only was the SNP orientation evident, but heterozygotes were also clear.