Methods for isolation of cell-free plasma DNA strongly affect DNA yield.

Author(s): Fleischhacker M, Schmidt B, Weickmann S, Fersching DM, Leszinski GS, Siegele B, Stötzer OJ, Nagel D, Holdenrieder S

Publication: Clin Chim Acta, 2011, Vol. 412, Page 2085-8

PubMed ID: 21861994 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, targeted gene and prior specimen transport the on quantification of cell-free DNA yields from plasma using real-time PCR. Plasma was obtained from pulmonary and oncology patients and stored at -80 degrees C in Berlin prior to the study. Plasma aliquots from each patient were thawed, pooled, and then realiquoted for shipping to Munich on dry ice or for additional storage in Berlin at -80 degrees C.

   Summary of Findings:

   The average DNA yield was 2-3 times higher in specimens extracted using the MagNA Pure isolation system than those extracted using NucleoSpin kit (p<0.0001), and average yields were 5-10 times higher using MagNA Pure than using QIAamp DNA blood Midi kit (p<0.0001). Real time qPCR based DNA quantification was twice as high when Beta-globin was the targeted gene than when determined by amplification of ERV or GAPDH (both p<0.001). Mailed specimens had decreased DNA yields when extracted using the MagNA PURE isolation system, and consequently, the Munich laboratory had smaller yields than the Berlin site using MagNA PURE, but not the other kits. (p=0.0126). The authors report higher DNA yields from cancer patients than from healthy controls, regardless of extraction method.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Benign
   • Neoplastic - Carcinoma
   • Neoplastic - Lymphoma
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Between site transportation method	Mailed Not transported
   Analyte Extraction and Purification	Analyte isolation method	QIAamp DNA blood Midi kit NucleoSpin kit MagNA Pure isolation system
   Real-time qPCR Specific	Targeted nucleic acid	ERV Beta-globin GAPDH

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