Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Evaluation of commercial kits for purification of circulating free DNA.

Author(s): Diefenbach RJ, Lee JH, Kefford RF, Rizos H

Publication: Cancer Genet, 2018, Vol. 228-229, Page 21-27

PubMed ID: 30553469 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared extraction kits and fragment size on the recovery of spiked-in DNA from plasma. Blood from four healthy individuals was immediately centrifuged at 800 x g for 15 min followed by centrifugation of the supernatant in a new tube at 1600 x g for 10 min. Plasma was immediately aliquoted and stored at -80˚C. Three 1 mL aliquots of plasma was spiked with 500 ng/ml of low molecular weight DNA ladder while the remaining aliquot was left unspiked. cfDNA was extracted from pooled plasma using each of the following kits: QIAamp Circulating nucleic acid kit, Plasma/Serum Cell-Free Circulating DNA Purification Midi kit, QIAamp MinElute ccfDNA Mini kit; Maxwell RSC ccfDNA Plasma kit, MagMAX cell-free DNA isolation kit, NextPrep-Mag cfDNA Isolation kit. Fragment size recovery was determined using an Experion automated electrophoresis system (running a DNA 1K analysis and comparing the purified amount to the input amount). The average recovery was based on the recovery of each individual fragment size.     

   Summary of Findings:

   Average recovery of spiked-in DNA was significantly higher when extraction was with the spin column-based QIAamp Circulating Nucleic Acid kit and Plasma/Serum Cell-Free Circulating DNA Purification Midi kit than when extracted using the magnetic bead-based kits (P<0.001, all). Among the magnetic bead-based kits, the QIAamp MinElute ccfDNA Mini kit resulted in the highest recovery rates and the NextPrep-Mag cfDNA Isolation kit the lowest recovery rates. All of the kits examined recovered spiked-in DNA fragments of 50-808 bp but none were able to efficiently recover the 25 bp fragment of spiked-in DNA. Consistent levels of recovery of fragments between 75-808 bp were observed for all kits with the exception of the NextPrep-Mag cfDNA Isolation kit which showed decreasing recovery with increasing fragment size of more than 200 bp.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp circulating nucleic acid kit Plasma/serum cell-free circulating DNA Purification midi kit QIAamp minElute ccfDNA mini kit Maxwell RSC ccfDNA plasma kit MagMAX cell-free DNA isolation kit NextPrep-Mag cfDNA isolation kit

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2. Study Purpose

   This study compared the recovery of endogenous and spiked-in cfDNA from plasma using two different extraction kits as measured by droplet digital PCR (ddPCR) amplification. Blood from five healthy individuals was immediately centrifuged at 800 x g for 15 min followed by centrifugation of the supernatant in a new tube at 1600 x g for 10 min. Plasma was immediately aliquoted and stored at -80˚C. Plasma was spiked with undiluted DNA extracted from the M229 cell line using the QIAamp Circulating Nucleic Acid kit or 10-fold diluted DNA. cfDNA was extracted using the QIAamp Circulating Nucleic Acid kit or the QIAamp MinElute ccfDNA Mini kit from plasma without spiked-in DNA, plasma with diluted M229 DNA, and plasma with undiluted M229 DNA. Purified endogenous cfDNA was quantified by droplet digital PCR amplification of NRAS. Spiked-in DNA was quantified by ddPCR amplification of mutant proto-oncogene B-Raf (BRAF).

   Summary of Findings:

   Significantly more endogenous NRAS cfDNA was obtained from plasma using the QIAamp Circulating Nucleic Acid kit than the QIAamp MinElute ccfDNA Mini kit (P=0.049). Similarly, there was higher recovery of the spiked-in BRAF mutated DNA when extraction was with the QIAamp Circulating Nucleic Acid kit, but the difference was only significant when the spiked-in DNA was undiluted.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Digital PCR Specific	Targeted nucleic acid	Endogenous NRAS Spiked-in BRAF Diluted spiked-in BRAF
   Analyte Extraction and Purification	Analyte isolation method	QIAamp circulating nucleic acid kit QIAamp minElute ccfDNA mini kit

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