Optimizing the proteinase K digest improves yield and integrity of DNA extracted from formalin-fixed,...

ABSTRACTAutomation is a requisite for laboratories performing large numbers of DNA extractions, but it can compromise DNA yield and/or integrity compared to manual extractions. We addressed this problem by optimizing the manual Proteinase K digest of our Chemagic MSM I robot, which performs automated DNA extractions from FFPE tissue. We sequentially optimized the volume of Proteinase K, digest time, temperature, thermomixing during digestion and the number of sections per extraction. We also evaluated using carrier RNA to enhance yield. DNA was quantified, purity-assessed and integrity-assayed at each step. The optimized protocol was validated using 24 FFPE blocks. Sequential optimization returned a 24-fold increase in DNA yield, but purity was slightly poorer. DNA integrity, assessed by DNA Integrity Numbers, qPCR and multiplex PCR, was also improved. A break point can be added between the Proteinase K digest and the DNA extraction (with samples stored at -80 °C). Carrier RNA increased DNA yield. The performance of a high-throughput robot that extracts DNA from FFPE with suboptimal efficiency can be radically improved, particularly by optimizing the Proteinase K digest step. Users should not assume the manufacturer's protocol is optimal.