Qiaamp ffpe tissue kit

DNA of each tissue sample was isolated from slides of 10 µm using a QIAamp® FFPE tissue kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The total DNA was quantified with a spectrophotometer at 260 nm. Next, 5 ng of the total DNA was amplified by real-time PCR using specific primers for 18S (forward 5′-GGA CAC GGA CAG GAT TGA CA-3′ and reverse 5′-ACC CAC GGA ATC GAG AAA GA-3′ for 18S) and the NADH dehydrogenase subunit 4 (forward 5′-CGT GAC TCC TAC CCC TCA CA-3′ and reverse 5′-ATC GGG TGA TGA TAG CCA AG-3′) on a LightCycler 480 System II (Basel, Switzerland, UChicago IGSB Next Generation Sequencing Core, RRID: SCR_011063) with 7.5 μL of Lightcycler^® 480 SYBR Green I Master (Basel, Switzerland), 0.5 μM of each set of primers, and 2.5 μL of the cDNA template. The PCR program was 95 °C for 5 min, and 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 12 s. A negative control was run in each assay. A ratio between the resulting PCR products (NADH dehydrogenase subunit 4/18S) was analyzed to evaluate the amount of mtDNA.

DNA extraction and mutation detection were performed using a modified protocol, as described previously [22 (link), 23 (link)]. Briefly, DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tumor tissue sections by the QIAamp FFPE Tissue Kit (Qiagen, Venlo, Netherlands). Following DNA extraction, an assay with RASKET KIT (MBL, Nagoya, Japan) was performed according to the manufacturer’s protocol. We simultaneously examined 146 KRAS mutations located in exon 2 codons 12 and 13, exon 3 codons 59 and 61, and exon 4 codon 117.