Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Selection of an optimal method for co-isolation of circulating DNA and miRNA from the plasma of pregnant women.

Author(s): Sedlackova T, Repiska G, Minarik G

Publication: Clin Chem Lab Med, 2014, Vol. 52, Page 1543-8

PubMed ID: 24815052 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of isolation method on the yield of tcDNA, cffDNA and miRNA from the plasma of pregnant women. Blood from 10 pregnant women carrying male fetuses was collected into K2EDTA tubes and stored at 4°C for <24h before centrifugation. Plasma was aliquoted and stored at -20°C. The vacuum step in the QIAamp Circulating Nucleic Acid Kit protocols was replaced with centrifugation at 2200 g for 1 min. All isolated circulating DNA and miRNA was stored at -80°C. tcDNA was quantified by TaqMan  amplification of the androgen receptor (AR) gene, and cffDNA was quantified through TaqMan amplification of the Y chromosomal marker DYS14. miRNA was quantified using miScript reverse transcription and amplification assays. Variability between replicates was investigated using 10 additional EDTA-plasma specimens.

   Summary of Findings:

   tcDNA, cffDNA and miRNA were successfully isolated using all three methods. When the QIAamp CNA Kit with the DNA protocol was used, yields of tcDNA were 1.3-fold and 2.1-fold higher and yields of cffDNA were 1.3 and 2.2-fold higher than when isolated using the QIAamp CNA kit with the RNA protocol or the miRcury kit, respectively. Importantly, only the increased cffDNA yield using the QIAamp CNA Kit with the DNA protocol rather than the miRcury kit was significant (p=0.0449). In contrast, the yields of miR-16 and miR-451 were 41 and 50-fold higher, respectively when isolated using the miRcury kit rather than the QIAamp CNA kit with the RNA protocol (p<0.0001, both). The same two miRNAs were 2448 and 2228-fold higher, respectively, using the miRcury kit rather than the QIAamp CNA kit with the DNA protocol (p<0.0001, both). When co-isolated using the miRcury kit, technical replicates displayed <1% variability in tcDNA and cffDNA quantification and <2% variability in miRNA quantification.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	miRCURY RNA Isolation Kit QIAamp Circulating Nucleic Acid Kit using a modification of the miRNA protocol QIAamp Circulating Nucleic Acid Kit using a modification of the circulating DNA protocol
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-16 miR-451
   Real-time qPCR Specific	Targeted nucleic acid	AR DYS14

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