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Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions.

Author(s): Lee EY, Lee EJ, Yoon H, Lee DH, Kim KH

Publication: Diagnostics (Basel), 2020, Vol. 10, Page

PubMed ID: 32325682 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the cell-free DNA (cfDNA) yield and DNA fragment size ratios among urine specimens extracted using four different extraction kits. cfDNA yield was also compared in urine stored for 3 months at -20°C versus -70°C with and without EDTA. First morning urine was collected from five healthy males and five healthy females and centrifuged at 200 x g for 10 min followed by centrifugation of the supernatant at 3000 x g for 20 min. The supernatants were aliquoted for extraction kit and storage condition experiments. For the comparison of extraction kits, 10 mM EDTA was added and specimens were frozen at -70°C briefly. Urine was thawed at 4°C, centrifuged at 3000 x g for 10 min, and DNA was extracted from 4-24 mL supernatant using the QIAamp Circulating Nucleic Acid Kit, MagMAX Cell-Free DNA Isolation Kit, Norgen Urine Cell-Free Circulating DNA Purification Midi Kit, and the Zymo Quick-DNA Urine Kit as detailed by the manufacturer. For the comparison of storage conditions, urine aliquots were stored with and without 10 mM EDTA at -70°C and -20°for 3 months. Urine was thawed at 4°C, centrifuged at 3000 x g for 10 min and DNA was extracted using the MagMAX Kit. cfDNA concentration and fragment distribution were assessed using the Agilent 2100 Bioanalyzer with High Sensitivity DNA chips. DNA of 50-300 bp was considered cfDNA while fragments greater than 1 kb were considered contamination by cellular DNA. No statistical analysis was performed.

   Summary of Findings:

   The highest yield of 50-300 bp cfDNA was obtained using the MagMAX Kit followed by the QIAamp CNA Kit. Of the 10 specimens, five yielded the most cfDNA in the 50-300 bp range using MagMAX and four using QIAamp CNA kit. The MagMAX Kit also yielded the most highly fragmented cfDNA of 50-100 bp in four specimens, with the Norgen Kit yielding the most highly fragmented cfDNA of 50-100 bp in the remaining six specimens. The average ratio of cfDNA to high molecular weight DNA was highest when extraction was performed using the Norgen Kit, indicating this kit is best at eliminating genomic contamination. Further, 6 of the 10 specimens had the highest ratio of cfDNA to high molecular weight DNA when extraction was with the Norgen Kit and three specimens with the MagMAX Kit. cfDNA yields were higher in specimens stored at -70°C than -20°C after 3 months. Addition of EDTA to the urine led to higher average cfDNA yields after 3 months at either temperature.

   Biospecimens

   • Bodily Fluid - Urine

   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 MagMAX Cell-Free DNA Isolation Kit Norgen Urine Cell-Free Circulating DNA Purification Midi Kit Zymo Quick-DNA Urine Kit
   Biospecimen Preservation	Type of fixation/preservation	EDTA Frozen
   Storage	Storage temperature	-20°C -80°C

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