Qiaseq dna quantimize kit

We extracted DNA from the FFPE samples using the Maxwell 16 FFPE Tissue LEV DNA Purification kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions and measured the concentration using the dsDNA BR Assay kit (Life Technologies, Carlsbad, CA, USA) in the Qubit 3.0 Fluorometer. We evaluated DNA quality by diluting the extracted DNA to 5-10 ng/µL and using it as a template for polymerase chain reaction (PCR). We conducted PCR using the QIAseq DNA QuantiMIZE kit (Qiagen, Hilden, Germany).

Whole blood DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen). DNA from FFPE tissues was obtained from 3 to 6 sections with 10-μm thickness, including more than 30% of the cancer area, or after microdissection using a microdissection laser (Leica Biosystems, Nussloch GmbH, Germany). One mL of residual LBC samples was centrifuged (× 12,000 g), and the pellets were resuspended in 95% ethanol and air-dried for 5 min. Both the FFPE sections and LBC pellets were then incubated with proteinase K (Promega, Madison, WI, USA) for 15 h at 70 °C, followed by a 1-h incubation at 98 °C in lysis buffer (Promega). After centrifugation (× 12,000 g), the supernatants were applied to the Maxwell RSC DNA FFPE kit and Maxwell 16 system (Promega). After measuring the extracted DNA concentration using the Qubit 3.0 dsDNA BR assay kit (Life Technologies, Grand Island, NY, USA), DNA qualities were monitored using the QIAseq DNA quantimize kit (Qiagen). A quality check score (QC score) less than 0.04 was considered as a high-quality DNA, according to our previous report [23 (link)].

After EUS-FNB samples were pathologically identified as PC using hematoxylin-eosin (HE) and immunohistochemical staining with the MIB-1 labeling index, DNA was extracted from FFPE and LBC samples using the Maxwell 16 FFPE Tissue LEV DNA Purification Kit (Promega, Madison, WI, USA) and from frozen samples using the Monarch^® Genomic DNA Purification Kit (New England BioLabs Inc., Ipswich, MA, USA). DNA from whole blood (control; QIAamp DNA Blood Mini Kit; Qiagen, Hilden, Germany) and EUS-FNB samples was extracted to identify tumor-specific gene mutations. The DNA concentration was measured using the Qubit 3.0. Fluorometer dsDNA BR assay kit (Life Technologies, Carlsbad, CA, USA), and the DNA quality was confirmed using the QIAseq DNA Quantimize kit (Qiagen). Real-time PCR was performed using the QIAseq DNA Quantimize kit, and Ct values were calculated from 100 and 200 bp amplicons. The quality check (QC) score (ΔCt200 − ΔCt100/200 − 100) was calculated to evaluate the DNA quality, with lower QC scores indicating less DNA degradation. If the QC score was less than 0.004 and the amplification was more than 0.5, the DNA was considered to be of high quality.

For DNA preparation from FFPE samples, we used the Maxwell 16 FFPE Tissue LEV DNA Purification kit (Promega) according to the manufacturer’s instructions. Thereafter, the concentration of DNA was measured using a Qubit 3.0 Fluorometer dsDNA BR Assay kit (Life Technologies), and DNA quality was monitored using the QIAseq DNA QuantiMIZE kit (QIAGEN). The extracted DNA was diluted to a concentration of 5–10 ng/μL as a template, and PCR was performed using the QIAseq DNA QuantiMIZE kit.