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

Xue, X.Y., Teare, M.D., Holen, I., Zhu, Y.M. and Woll, P.J. (2009) Optimizing the yield and utility of circulating cell-free DNA from plasma and serum. Clinica Chimica Acta, 404 (2). pp. 100-104. ISSN 0009-8981

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Abstract

Background: Cell-free DNA (CFDNA) in the plasma/serum of patients with cancer demonstrates tumour-associated genetic alterations. offering possibilities for diagnosis, prognostication and disease monitoring. There is wide variation in the reported levels of CFDNA, associated with different methods used to collect, process and analyze blood samples. We therefore evaluated different aspects of laboratory protocols for the processing and purification of CFDNA in clinical studies.

Methods: We evaluated and compared the QlAamp kit and a Triton/Heat/Phenol protocol (THP) for CFDNA purification. Total CFDNA was quantified by PicoGreen assay and SYBR-Green real-time PCR assay was used to amplify specific genes to estimate the efficiency of different protocols.

Results: The efficiency of DNA extraction was 18.6% using the standard QlAamp protocol and 38.7% using the THP method (p<0.0001, unpaired t-test). A modified QlAamp protocol that included a proteinase incubation stage and elution volumes up to 300 mu l increased DNA yields, but was not as good as the THP method.

Conclusions: Blood samples should be kept at/or below room temperature (18 degrees C-22 degrees C) for no more than 2 h before plasma separation by double-spin. Because of its higher efficiency, low-cost and good-quality products, the THP protocol is preferred for extraction of CFDNA.

Item Type: Article
Copyright, Publisher and Additional Information: © 2009 Elsevier. This is an author produced version of a paper subsequently published in Clinica Chimica Acta. Uploaded in accordance with the publisher's self-archiving policy.
Keywords: Cell-free DNA; Plasma DNA; Serum DNA; Methodology; DNA extraction protocol; Analysis
Institution: The University of Sheffield
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > School of Medicine (Sheffield) > Department of Infection & Immunity
The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > School of Medicine (Sheffield) > Division of Genomic Medicine (Sheffield) > Department of Oncology (Sheffield)
Depositing User: Miss Anthea Tucker
Date Deposited: 24 Jul 2009 11:19
Last Modified: 04 Jun 2014 14:28
Published Version: http://dx.doi.org/10.1016/j.cca.2009.02.018
Status: Published
Publisher: Elsevier
Refereed: Yes
Identification Number: 10.1016/j.cca.2009.02.018
URI: http://eprints.whiterose.ac.uk/id/eprint/9014

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