Towards standardisation of cell-free DNA measurement in plasma: Controls for extraction efficiency, fragment size bias and quantification

252Citations
Citations of this article
478Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp® circulating nucleic acid (CNA) kit, NucleoSpin® Plasma XS (NS) kit and FitAmp™ plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity. [Figure not available: see fulltext.]

Cite

CITATION STYLE

APA

Devonshire, A. S., Whale, A. S., Gutteridge, A., Jones, G., Cowen, S., Foy, C. A., & Huggett, J. F. (2014). Towards standardisation of cell-free DNA measurement in plasma: Controls for extraction efficiency, fragment size bias and quantification. Analytical and Bioanalytical Chemistry, 406(26), 6499–6512. https://doi.org/10.1007/s00216-014-7835-3

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free