Automated Capillary Electrophoresis Based Fragment Size Distribution Analysis of cfDNA: A DNA Microchip-based Method to Study Size Distribution of DNA Fragments Isolated from Biofluids

Abstract

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

In this video, we analyze the size distribution of cell-free DNA or cfDNA fragments using automated capillary electrophoresis. This technique helps gain insights into the profile of DNA fragments released by cancer cells during apoptosis.

Protocol

1. DNA Fragment Size Distribution of cfDNA by Fragment Analyzer The step before initiating the procedure: Equilibrate the DNA dye concentrate and DNA gel matrix to room temperature for 30 min. Preparation of the gel-dye mix NOTE: Handle solutions with caution as DMSO is known to facilitate the entry of organic molecules into tissues. Thoroughly thaw the DMSO by vortexing the DNA dye concentrate vial for 10 s. Pipette out 15 µL of this concent…

Materials

2100 Bioanalyzer Instrument	Agilent Technologies, Inc.	G2939BA	The 2100 Bioanalyzer system is an established automated electrophoresis tool for the sample quality control of biomolecules.
Adjustable Clip for Priming Station	Agilent Technologies, Inc.	5042-1398	Used in combination with syringe to apply defined pressure for chip priming.
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 cf-DNA, circulating tumor DNA, cf-RNA and circulating tumor cells in human whole blood samples during storage and shipping
Chip Priming Station	Agilent Technologies, Inc.	5065-4401	Used to load gel matrix into a chip with a syringe provided with each assay kit— used for RNA, DNA, and protein assays. Includes priming station, stop watch, and 1 syringe clip
Electrode Cleaner Kit	Agilent Technologies, Inc.	5065-9951	Prevents cross-contamination. Removes bacterial or protein contaminants from electrodes.
Filters for Gel Matrix	Agilent Technologies, Inc.	185-5990	Used for proper mixing of DNA dye concentrate and DNA gel matrix
IKA Basic Chip Vortex	IKA-Werke GmbH & Co. KG	MS-3-S36	Used for proper mixing of DNA ladder and DNA sample on Bioanalyzer assay chips
50 ml centrifuge tubes
Ethanol (96–100%)
Heating block or similar at 56 °C (capable of holding 2 ml collection tubes)
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