Four FFPE tissues types were obtained from Biochain Institute, Inc. (Newark, CA): human lung tumor (Cat.# T2235152-D01), human breast tumor (Cat.# T2235086-D01), human colon tumor (Cat.# T2235090-D01) and human adult normal heart (Cat.# T2234122). DNA was purified from single 5µm FFPE tissue sections using three methods: 1) The ReliaPrep™ FFPE gDNA Miniprep System was used according to the standard protocol (1) , which includes a decross-linking step consisting of an 80°C incubation for 1 hour, 2) The ReliaPrep™ FFPE gDNA Miniprep System was used with a modified protocol, where the incubation time for decross-linking was increased to 4 hours and 3) The QIAamp® DNA FFPE Tissue Kit was used according to the standard protocol (2) , which includes a decross-linking step consisting of a 90°C incubation for 1 hour. For all methods, DNA was eluted in 30µl, with 30µl of eluate recovered.
DNA recovery was determined by qPCR analysis using primer sets that produced amplicons of varying sizes. As DNA purified from FFPE tissue is often degraded and highly fragmented, we chose amplicon sizes of less than 150bp in length for more accurate DNA quantitation. Amplification of larger amplicons will likely underestimate the available DNA. The GoTaq® qPCR Master Mix (dsDNA-binding dye-based qPCR, Cat.# A6001) was used with two primer sets specific to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), resulting in 100bp and 300bp amplicons. Two microliters of DNA was added in a 25µl total reaction volume. The GoTaq® Probe qPCR Master Mix (fluorogenic probe-based qPCR, Cat.# A6101) was used with two primer sets specific to RNaseP and telomerase reverse transcriptase (TERT), resulting in 102bp and 164bp amplicons, respectively. Two microliters of DNA was added in a 20µl total reaction volume. For both master mixes, DNA was quantified using the Plexor® HY Male Genomic DNA Standard at amounts ranging from 100ng to 10pg. All samples were analyzed using the BioRad CFX 96 Real-Time PCR System.