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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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

Weijie Chen1 / Fengfeng Cai1 / Bei Zhang1 / 1

1Laboratory for Gynecological Oncology, Department of Biomedicine, University Women’s Hospital, University of Basel, Basel, Switzerland

Corresponding author: Xiao Yan Zhong, Laboratory for Gynecological Oncology, Department of Biomedicine, University Women’s Hospital, University of Basel, Hebelstr, 20, 4031 Basel, Switzerland Phone: +41 612 659 248, Fax: +41 612 659 399

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM). Volume 50, Issue 2, Pages 261–265, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/cclm.2011.773, October 2011

Publication History

Received:
2011-07-07
Accepted:
2011-10-03
Published Online:
2011-10-31

This article offers supplementary material which is provided at the end of the article.

Abstract

Background: Circulating cell-free (ccf) DNA in blood has been suggested as a potential biomarker in many conditions regarding early diagnosis and prognosis. However, misdiagnosis can result due to the limited DNA resources in Biobank’s plasma samples or insufficient DNA targets from a predominant DNA background in genetic tests. This study explored several strategies for an efficient DNA extraction to increase DNA amount from limited plasma input.

Methods: Ccf plasma DNA was extracted with three different methods, a phenol-chloroform-isoamylalcohol (PCI) method, a High Pure PCR Template Preparation Kit method and a method used for single cell PCR in this group. Subsequently, the total DNA was measured by Nanodrop and the genome equivalents (GE) of the GAPDH housekeeping gene and MTATP 8 gene were measured using a multiplex real-time quantitative PCR for the quantitative assessment of nDNA and mtDNA.

Results: Instead of 400–800 μL (routine input in the laboratory), 50 μLof plasma input enabled the extraction of ccf DNA sufficient for quantitative analysis. Using the PCI method and the kit method, both nDNA and mtDNA could be successfully detected in plasma samples, but nDNA extracted using protocol for single cell PCR was not detectable in 25% of plasma samples. In comparison to the other two methods, the PCI method showed lower DNA purity, but higher concentrations and more GE of nDNA and mtDNA.

Conclusions: The PCI method was more efficient than the other two methods in the extraction of ccf DNA in plasma. Limited plasma is available for ccf DNA analysis.

Keywords: circulating cell-free DNA; mitochondrial DNA; nuclear DNA

Supplementary Article Materials

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[1]
Bernhard Ralla, Carsten Stephan, Sebastian Meller, Dimo Dietrich, Glen Kristiansen, and Klaus Jung
Critical Reviews in Clinical Laboratory Sciences, 2014, Volume 51, Number 4, Page 200

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