Isolation of Circulating Cell-free DNA (cfDNA): A Silica-based Membrane Procedure to Extract cfDNA from Blood Plasma of Cancer Patients
Published: April 30, 2023
Abstract
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. Preparation of buffers and reagents CAUTION: Do …
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
Etiketler
Bu Makaleden Alıntı Yapın
Isolation of Circulating Cell-free DNA (cfDNA): A Silica-based Membrane Procedure to Extract cfDNA from Blood Plasma of Cancer Patients. J. Vis. Exp. (Pending Publication), e20671, doi: (2023).