Real time pcr mx3005p

Germline DNAs from collected peripheral blood mononuclear cells were isolated using a QIAamp DNA mini kit (Qiagen, Santa Clarita, CA, USA). Circulating DNAs were extracted from 1–5 mL of plasma using a QIAamp Circulating Nucleic Acid Kit (Qiagen). DNA concentration and purity were assessed by a Picogreen fluorescence assay using a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY, USA) with a Qubit dsDNA HS Assay Kit and a BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). DNA concentration and purity were quantified using a Nanodrop 8000 UV-Vis spectrometer (Thermo Fisher Scientific) and a Picogreen fluorescence assay using a Qubit 2.0 Fluorometer (Life Technologies). The fragment size distribution was measured using a 2200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA, USA) and real-time PCR Mx3005p (Agilent Technologies) according to the manufacturer’s instructions.

Germline genomic DNA from PBLs was purified by QIAamp DNA mini kits (Qiagen, Santa Clarita, CA, USA). Circulating cfDNA was extracted from 2 to 5 mL plasma using QIAamp Circulating Nucleic Acid kits (Qiagen). DNA concentration and purity were assessed by Nanodrop 8000 UV-Vis spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit 2.0 fluorometer using Picogreen fluorescence assays (Life Technologies, Grand Island, NY, USA). Fragment size distribution was estimated using a 2200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA, USA) and real-time PCR Mx3005p (Agilent Technologies) according to the manufacturer’s manual.

For the evaluation of analytical performance, purified DNA from 20 normal HapMap cell lines (Supplementary Table S1) were purchased from the Coriell Institute (http://ccr.coriell.org/). For indel validation, 10 immortalized tumor cell lines (Supplementary Table S2) were purchased from ATCC (American Type Culture Collection, http://www.atcc.org). Genomic DNA was extracted from cell lines using QIAamp DNA Mini Kits (Qiagen, Valencia, CA, USA). In the case of FFPE clinical samples, DNA was extracted from 40 μm of unstained FFPE sections, typically 4 × 10 μm sections, using a Promega Maxwell 16 CSC DNA FFPE kit with an automation instrument (Promega, Fitchburg, WI, USA). DNA concentration and purity were assessed by a Nanodrop 8000 UV-Vis spectrometer (Thermo Scientific, Waltham, MA, USA) and a Picogreen fluorescence assay using a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY, USA). The fragment size distribution, indicating the degree of DNA degradation, was measured using a 2200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA, USA) and real-time PCR Mx3005p (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s manual.