Qiaamp dna ffpe tissue kit 50

Genomic DNA (gDNA) was isolated from FFPEs using the QIAamp^® DNA FFPE Tissue Kit 50 (Qiagen^®, Germany) according to the manufacturer instructions. cfDNA from plasma (2mL) was extracted using the QIAamp^® Circulating Nucleic Acid kit 50 (Qiagen^®, Germany), according to the manufacturer's instructions. DNA concentration was determined in the Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, USA).

Genomic DNA (gDNA) was isolated from FFPEs using the QIAamp^® DNA FFPE Tissue Kit 50 (QIAGEN^®, Hilden, Germany), according to manufacturer instructions. For the plasma, 2 × 5 mL peripheral blood samples were collected in EDTA tubes at the time point of diagnosis, before tumor surgery and before the application of therapeutic substances with an S-Monovette. Blood was centrifuged at 1500× g for 10 min, and the plasma was stored at −80 °C until further analysis. Additionally, cfDNA was extracted from plasma (2 mL) using the QIAamp^® Circulating Nucleic Acid kit 50 (QIAGEN^®, Hilden, Germany), according to the manufacturer’s instructions. The DNA concentration was determined in the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as previously described [35 (link)].

FFPEs: FFPEs containing >60% tumor cells were used for genomic DNA (gDNA) extraction. gDNA was isolated from FFPEs with using the QIAamp^® DNA FFPE Tissue Kit 50 (Qiagen^®, Hilden, Germany), according to the manufacturer’s instructions. The DNA concentration was determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, NC, USA).
Plasma: 10 mL of peripheral blood in EDTA were used within 2–4 h to isolate plasma via centrifugation at 530× g for 10 min. Following a second centrifugation at 2000× g for 10 min, plasma was transferred into 2 mL tubes and stored at −70 °C until use. cfDNA was further isolated using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany), as previously described [35 (link)].
In all samples, cfDNA quality was checked prior to PCR using a previously described protocol [36 (link)]. Serial dilutions of a wild-type sample with a known DNA concentration (Human Reference DNA Female, Agilent Technologies, Santa Clara, CA, USA), prepared via serial 10-fold dilution in concentrations ranging from 200 ng/μL down to 0.5 ng/μL, were used to generate a standard curve for the quantification of the gDNA concentration in all cfDNA samples using a LightCycler z480 (Roche).