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

1. The step before initiating the procedure: Equilibrate the DNA dye concentrate and DNA gel matrix to room temperature for 30 min.
2. Preparation of the gel-dye mix
   NOTE: Handle solutions with caution as DMSO is known to facilitate the entry of organic molecules into tissues.
   1. Thoroughly thaw the DMSO by vortexing the DNA dye concentrate vial for 10 s. Pipette out 15 µL of this concentrate into a DNA gel matrix vial and store at 4 °C in the dark.
   2. Again, vortex the capped vial for 10 s until the mixing of the gel and dye is visualized.
   3. Pour the mix on a spin filter to the top receptacle.
   4. Microcentrifuge the spin filter at 2,240 x g ± 20% for 10 min at room temperature.
   5. Label the prepared gel-dye in the tube and discard the filter, as per good laboratory practices. Label the tube and record the date of preparation.
      NOTE: Discard the filtrate as per good laboratory practices. The gel-dye mix can be used for 5 High Sensitivity (HS) DNA chips. If unused for more than 1 h, store at 4 °C. Storage in the dark is possible for up to 6 weeks.
3. To load the gel-dye mix, ensure the position of the base plate of the chip priming station and adjust the clip at the lowest position.
   1. Equilibrate the gel-dye mix to room temperature for 30 min, while monitoring light exposure.
   2. Take a new HS DNA chip from a sealed bag and place it on the chip priming station, then remove 9.0 µL of the gel-dye mix and dispense it at the bottom of the chip well, marked as 'G'.
      NOTE: Draw up the gel-dye mix, avoiding particles that may accumulate at the bottom of the vial. While dispensing the gel-dye mix into the HS DNA chip well, insert the tip of the pipette completely to prevent the formation of large air bubbles. Moreover, touching the pipette at the edges of the well will produce poor results.
   3. Position the plunger at 1 mL and close the chip priming station. Ensure the lock of the latch clicks and set the timer to 60 s, then press the plunger down until it is held by the clip, and exactly after 60 s, release the plunger with the clip-release mechanism.
   4. When the plunger retreats at least to the 0.3 mL mark, wait for 5 s, and then slowly pull back to the 1 mL position, then open the chip priming station and again remove 9.0 µL of the gel-dye mix and dispense at the bottom of the HS DNA chip well, marked as 'G'.
4. To load the DNA marker, dispense 5 µL of the DNA marker into the well, marked with the ladder symbol. Repeat the procedure for all the 11 sample wells.
5. To load the ladder and samples, dispense 1 µL of the DNA ladder in the well, marked with the ladder symbol, and then add 1 µL of sample (used wells) or 1 µL of marker (unused wells) in all the 11 sample wells.
6. Vortex the HS DNA chip for 60 s at 2,400 rpm by placing the chip horizontally in the adapter. Ensure that the bulge that fixes the HS DNA chip is not damaged during vortexing.
7. To insert the HS DNA chip in the fragment analyzer instrument, open the lid and ensure that the electrode cartridge is properly inserted, and the chip selector is positioned to 'dsHigh Sensitivity DNA' in the fragment analyzer instrument.
8. Carefully mount the HS DNA chip into the receptacle, which fits one way only, then close the lid by ensuring that the electrode cartridge fits exactly into the wells of the HS DNA chip.
9. The display on the fragment analyzer software screen indicates the inserted HS DNA chip and the closed lid through the chip icon at the top left of the screen.
10. To initiate the HS DNA chip run, select the dsDNA High Sensitivity Assay from the 'Assay' menu on the instrument screen, then properly fill the table of sample names by feeding information such as sample names and comments and start the chip run by clicking the 'Start' button at the upper right of the screen.
11. Electrode cleaning after an HS DNA chip run: Immediately remove the used HS DNA chip as soon as the assay is completed and dispose of it according to good laboratory practices. Perform the following procedure to ensure the electrodes are clean, without leftover residues from the previous assay.
    1. Fill slowly 350 µL of deionized analysis-grade water into one of the electrode cleaner wells and place the electrode cleaner in the fragment analyzer instrument by opening the lid and then close the lid and wait for about 10 s.
    2. Remove the electrode cleaner by opening the lid and wait for another 10s for the water on the electrodes to evaporate before closing the lid.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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