Optimizing the yield and utility of circulating cell-free DNA from plasma and serum.

Author(s): Xue X, Teare MD, Holen I, Zhu YM, Woll PJ

Publication: Clin Chim Acta, 2009, Vol. 404, Page 100-4

PubMed ID: 19281804 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 and delayed centrifugation of whole blood on the yield of cell-free DNA from blood. Commercially produced serum was spiked with reference DNA and linearized bcl-2. Plasma was stored at -70 degrees C until analysis. DNA extracted from plasma using QIAamp Blood DNA kit was predigested with the included protease or 400 mg/L proteinase K at 37 degrees C or 50 degrees C for 0, 1 or 2 h. The THP protocol involved lysis with Triton X-100, denaturation at 98 degrees C for 5 min, and a standard extraction with phenol/chloroform/isoamyl alcohol followed by sodium acetate precipitation.

   Summary of Findings:

   The recovery of spiked DNA from serum was increased when specimens were preincubated with proteinase K at 37 or 50 degrees C for 1 h compared to when no preincubation step was included (p=0.002-0.003) and when proteinase K was used instead of the QIAamp protease (p=0.040), but the authors report no effect of increasing the preincubation time from 1 h to 2 h. The recovery of spiked DNA was increased when the total elution volume used in the QIAamp protocol was increased from 50 uL to 300 uL, but no further increase in yield was observed when the total volume was increased to 500 uL, and the authors report there was no effect of prewarming the elution buffer. When DNA extraction was performed with THP, the GAPDH gene was amplifiable in all 15 pooled serum specimens, but when DNA was extracted using QIAamp, only 3 of 15 pooled serum specimens generated DNA that was amplifiable. Storing whole blood in EDTA vacutainers at room temperature or on ice for up to 2 h before separation of plasma had no effect on cell-free DNA yield, but when blood was stored for more than 2 h at either temperature, the yield of cell-free DNA increased. The increase in cell-free DNA yield with storage was not significantly different between specimens stored on ice or at room temperature.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 h 1 h 2 h 4 h 7 h 25 h
   Storage	Storage temperature	18-20 degrees C 0 degrees C (on ice)
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Analyte Extraction and Purification	Analyte isolation method	QIAamp Triton X/heat/phenol chloroform
   Analyte Extraction and Purification	Protein digestion	Preincubation with QIAamp protease Preincubation with 400 mg/L proteinase K 37 degrees C 50 degrees C 0 h 1 h 2 h
   Analyte Extraction and Purification	Rehydration of dried sample/specimen	50 uL Two 50 uL elutions Two 100 uL elutions A 100 uL and a 200 uL elution Two 200 uL elutions A 300 uL and a 200 uL elution Room temperature elution buffer 37 degrees C elution buffer 50 degrees C elution buffer
   Real-time qPCR Specific	Targeted nucleic acid	GAPDH
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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