Plasma ctDNA Extraction and Quantification

For plasma samples, 14 mL of peripheral blood was collected in EDTA-coated tubes before and 4 weeks after afatinib treatment, and upon disease progression. Plasma was separated by centrifugation at 1000 rpm for 15 min within 2 h of sample collection and stored at −80 °C until DNA extraction. Plasma ctDNA was purified using an AVENIO cfDNA isolation kit (Roche Diagnostics), and the quality and quantity of the DNA were verified using the NanoDrop 2000 device (Thermo Scientific) and PicoGreen dsDNA assay kit (Life Technologies) according to previous study^{11 (link)}. The extracted ctDNA was stored at −80 °C until the analysis.

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Ishii H., Azuma K., Sakai K., Naito Y., Matsuo N., Tokito T., Yamada K., Hoshino T, & Nishio K. (2020). Determination of Somatic Mutations and Tumor Mutation Burden in Plasma by CAPP-Seq during Afatinib Treatment in NSCLC Patients Resistance to Osimertinib. Scientific Reports, 10, 691.

Publication 2020

AVENIO cfDNA isolation kit NanoDrop 2000 device PicoGreen dsDNA assay kit

Afatinib Assay Blood Centrifugation Cfdna Device Diagnostics Disease progression Dsdna Edta Isolation Picogreen Plasma Sample collection

Corresponding Organization :

Other organizations : Kurume University, Kindai University, Koga Hospital

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Variable analysis

independent variables

Afatinib treatment

dependent variables

Plasma ctDNA

control variables

Time of sample collection (before treatment, 4 weeks after treatment, and upon disease progression)
Sample collection method (14 mL of peripheral blood collected in EDTA-coated tubes)
Plasma separation method (centrifugation at 1000 rpm for 15 min within 2 h of sample collection)
Sample storage (-80 °C until DNA extraction)
DNA extraction method (AVENIO cfDNA isolation kit)
DNA quality and quantity assessment (NanoDrop 2000 device and PicoGreen dsDNA assay kit)

controls

No positive or negative controls were explicitly mentioned in the protocol.

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