Strategies of reducing input sample volume for extracting circulating cell-free nuclear DNA and mitochondrial DNA in plasma.

Author(s): Chen W, Cai F, Zhang B, Zhong XY

Publication: Clin Chem Lab Med, 2012, Vol. 50, Page 261-5

PubMed ID: 22040238 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 DNA extraction method on the quantity and quality of ccf DNA, including nDNA and mtDNA, obtained from plasma. K3EDTA-plasma was stored at -80 degrees C prior to analysis. Ccf DNA obtained by the single cell PCR method was not evaluated by nanodrop, but amplified directly by real-time qPCR.

   Summary of Findings:

   The 260/280 ratio of DNA extracted by the PCI method was lower than that of DNA extracted by the High Pure kit, however, the PCI method yielded a higher DNA concentration after DNA extracted from 50 uL of plasma was dissolved in 12.5 uL tris-EDTA buffer compared to the High Pure kit which extracted DNA from 200 uL of plasma and required 50 uL tris-EDTA buffer for column washing. Both nDNA and mtDNA were detected using each of these two extraction methods. Using the protocol for single cell PCR (5 uL of plasma), mtDNA was detected in all samples, while nDNA was only detected in 75% of single cell PCR samples. The GE of nDNA and mtDNA were 5.5-fold and 2-fold higher, respectively, when DNA was extracted by the PCI method than by the High Pure kit, although this was not statistically significant. Further, the cycle threshold (Ct) values for mtDNA extracted by PCI were lower than the Ct values using DNA extracted by the High Pure kit.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	PCI High Pure PCR Template Preparation kit Single cell PCR method
   Biospecimen Aliquots and Components	Aliquot size/volume	5 uL 50 uL 200 uL
   Real-time qPCR Specific	Targeted nucleic acid	GAPDH Mitochondrial MTATP 8

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