Isolation of Circulating Cell-free DNA (cfDNA): A Silica-based Membrane Procedure to Extract cfDNA from Blood Plasma of Cancer Patients

Samenvatting

Source: Palande, V. et al. Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients. J. Vis. Exp. (2020)

This video describes a silica-based membrane procedure to isolate circulating cell-free DNA or cfDNA from human plasma sample. The cfDNA is a promising biomarker for cancer diagnosis and assessment. 

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Purification of Circulating cfDNA from 1 mL of Plasma

NOTE: This step is performed with a commercial kit (see Table of Materials). All buffers are provided with the kit.

1. Preparation of buffers and reagents
   CAUTION: Do not add acidic solutions or bleach directly to the sample preparation waste. Guanidine salts present in Lysis buffer, Binding buffer, and Wash Buffer-....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Agilent High Sensitivity DNA Kit	Agilent Technologies, Inc.	5067-4626	The High Sensitivity DNA assays are often used for sample quality control for next-generation sequencing libraries.
cf-DNA/cf-RNA Preservative Tubes	Norgen Biotek Corp.	63950	Norgen's cf-DNA/cf-RNA Preservative Tubes are closed, evacuated plastic tubes for the collection and the preservation of cfDNA, circulating tumor DNA, cf-RNA and circulating tumor cells in human whole blood samples during storage and shipping
QIAamp circulating nucleic acid kit	Qiagen	55114	The QIAamp Circulating Nucleic Acid Kit enables efficient purification of these circulating nucleic acids from human plasma or serum and other cell-free body fluids.
QIAvac 24 Plus vacuum manifold	Qiagen	19413	The QIAvac 24 Plus vacuum manifold is designed for vacuum processing of QIAGEN columns in parallel.
QIAvac Connecting System	Qiagen	19419	In combination with the QIAvac Connecting System, the QIAvac 24 Plus vacuum manifold can be used as a flow-through system. The sample flow-through, containing possibly infectious material, is collected in a separate waste bottle.
QIAvac Connecting System	Qiagen	19419	In combination with the QIAvac Connecting System, the QIAvac 24 Plus vacuum manifold can be used as a flow-through system. The sample flow-through, containing possibly infectious material, is collected in a separate waste bottle.
Miscellaneous
50 mL centrifuge tubes
Crushed ice
Ethanol (96–100%)
Heating block or similar at 56 °C (capable of holding 2 mL collection tubes)
Isopropanol (100%)
Microcentrifuge
Phosphate-buffered saline (PBS)
Pipettes (adjustable)
Sterile pipette tips (pipette tips with aerosol barriers are recommended to help prevent cross-contamination)
Water bath or heating block capable of holding 50 mL centrifuge tubes at 60 °C