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Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine.

Author(s): Oreskovic A, Brault ND, Panpradist N, Lai JJ, Lutz BR

Publication: J Mol Diagn, 2019, Vol. 21, Page 1067-1078

PubMed ID: 31442674 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effect of extraction kits on recovery of different size fragments of DNA from urine, recovery of low concentration spiked-in DNA, and effects on PCR inhibition. The effect of pH, salt concentration, and urine DNA concentration on the recovery of spiked in DNA from buffer using each kit was also examined. Urine from five healthy volunteers were pooled, preserved with 10 mmol/L EDTA, and stored at -80°C. DNA was extracted from urine using Q Sepharose anion exchange resin, Norgen Urine Cell-free Circulating DNA Purification Mini Kit, QIAamp Circulating Nucleic Acid Kit, MagMAX Cell-free DNA Isolation Kit, after mixing with guanidinium thiocyanate using Wizard Minipreps DNA Purification Resin, or by hybridization capture. Different size regions (25, 40, 80, and 150 bp) of IS6110 were quantified by real-time PCR and inhibitory effects were investigated by adding in varying amounts of cfDNA extracted from urine.

   Summary of Findings:

   While the hybridization capture method recovered >75% of the spiked-in synthetic DNA of each fragment size, the Norgen Kit recovered 72% of the 25 nt fragment but less than 50% of all other fragments, the Q Sepharose method recovered 63-75% of the 40-150 nt fragments but only 9% of the 25 nt fragment, the MagMAX Kit recovered 66% of the 150 nt fragment but <50% of all other fragments, and the Wizard/GuSCN and QIAamp Kit recovered <20% of the DNA of each size. Further,  PCR was positive (at least one replicate) for all urine specimens spiked with low concentrations (10 copies/mL) of the 40 nt fragment when DNA was isolated with hybridization capture, Q sepharose, or Norgen, but the signal was weaker in specimens extracted using Norgen and specimens were PCR positive in every reaction only when DNA was isolated by hybridization capture. Further investigation using buffer instead of urine found that while most of the methods were robust to pH, salt concentration, and urine DNA concentration, recovery of DNA using the Wizard/GuSCN method decreased as pH increased above 6 and improved with higher background DNA concentrations. Importantly, cfDNA extracted from urine using hybridization capture, Wizard/GuSCN, Q Sepharose, and QIAamp showed no inhibitory effect on PCR when the reaction contained <40% extracted cfDNA, but cfDNA extracted using MagMAX showed PCR inhibition when the reaction contained ≥20% extracted DNA and cfDNA extracted using Norgen had an inhibitory effect on PCR regardless of the amount added.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Biospecimen components	10 copies/mL spiked in DNA 100,000 copies/mL spiked in DNA
   Real-time qPCR Specific	Length of gene fragment	25 nt 40 nt 80 nt 150 nt
   Analyte Extraction and Purification	Analyte isolation method	Wizard resin/guanidinium thiocyanate Q Sepharose Norgen QIAamp MagMAX

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