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EPICENTRE Forum 5 (3) Isolation of DNA from Paraffin-Embedded Tissue Using the MasterPure™ Complete DNA and RNA Purification Kit Introduction One of the most active research areas in molecular pathology is retrospective studies on archival tissue samples. However, isolating high-quality genomic DNA from formalin-fixed, paraffin-embedded tissue can be difficult because only minimal amounts of intact DNA may be present in the sample.1 Because of this, analysis of the recovered DNA is generally limited to PCR, and amplification of small target sequences (300 bp or less) is most successful. The MasterPure™ Complete DNA and RNA Purification Kit was designed to isolate DNA and RNA from a variety of sources, including samples containing small amounts of nucleic acid. Here, we provide a protocol for isolating PCR-ready DNA from paraffin-embedded tissue using the MasterPure Complete Kit. We isolate genomic DNA from a biopsy specimen and show that the DNA is suitable as a template for PCR by amplifying a region of the Factor V gene. Methods and Results DNA isolation from breast cancer tissue paraffin sections The protocol for treatment of formalin-fixed, paraffin-embedded tissue samples prior to purification using the MasterPure Complete DNA and RNA Purification Kit is summarized in Table 1. The isolation of genomic DNA from a breast cancer tissue section was performed following these guidelines. Specifically, DNA was isolated from 0.02 g of 35 µm thick paraffin-embedded samples. (Thin paraffin sections allow the best recovery and quickest extraction times.) Five milliliters of xylene were added to the tissue and the sample was incubated for 10 minutes to extract the paraffin. The xylene was poured off and the extraction was repeated. Five milliliters of 100% ethanol were then added and the sample was incubated for 10 minutes. The ethanol was decanted and the ethanol extraction was repeated. The last traces of ethanol were removed by aspiration and the tissue was resuspended in 300 µl of Tissue and Cell Lysis Buffer 1 containing 1 µl of 50 mg/ml Proteinase K. The sample was incubated at 37°C for 30 minutes. The sample was then treated with 150 µl of Protein Precipitation Reagent, mixed by vortexing, and centrifuged for 10 minutes in a microcentrifuge. The DNA-containing supernatant was transferred to a clean microcentrifuge tube and 500 µl of isopropanol were added. The tube was inverted 30 times and then centrifuged for 10 minutes at 4°C in a microcentrifuge. The nucleic acid pellet was then washed twice with 70% ethanol and resuspended in 50 µl of TE buffer.
Factor V amplification using the isolated DNA One microliter of the human genomic DNA sample (2% of the total isolated) was used to amplify a 267 bp region of the Factor V gene. The sequences of the primers used were: 5'-TGTTATCACTGGTGCTAA-3' and 5'-TGCCCAAGTGCTTAACAAGACCA-3'. The 50 µl reaction contained 1X MasterAmp™ PCR Optimization Kit PreMix B (1X PCR buffer, 2.5 mM MgCl2, 200 µM each dNTP) (Epicentre), 50 pmoles of each Factor V primer, 1.25 units of MasterAmp AmpliTherm™ DNA Polymerase (Epicentre), and 1 µl of the genomic DNA template. Forty cycles of amplification were performed with the following profile: 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds. Five microliters of the sample were separated on a 2% agarose gel and the gel was stained with ethidium bromide. Figure 1 shows that the 267 bp fragment was easily amplified.
Summary The MasterPure Complete DNA and RNA Purification Kit can be used to isolate PCR-ready DNA from formalin-fixed, paraffin-embedded tissue samples using the protocol modifications described above. This protocol may be useful for isolating human DNA for applications such as retrospective amplification studies on archival tissue samples.
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