Title: Relative Quantification of Genomic DNA Fragments Extracted from a Biological Tissue
Authors: CORBISIER Philippe
Publisher: Publications Office of the European Union
Publication Year: 2009
JRC N°: JRC56358
ISBN: 978-92-79-14848-4
ISSN: 1018-5593
Other Identifiers: EUR 24143 EN
URI: http://publications.jrc.ec.europa.eu/repository/handle/JRC56358
DOI: 10.2787/22361
Type: EUR - Scientific and Technical Research Reports
Abstract: The provision of traceable standards to the biological community is an area of active research in many NMIs.The quantification of the relative amount of DNA sequences extracted from a biological tissue remains a complex analytical procedure and relies on the availability of such standards. Real-time PCR is currently the most applied measurement method to identify and quantify DNA sequences. Several NMIs were able to demonstrate their ability to use this technology to quantify a defined plasmid DNA using the same plasmid DNA as a calibrant (CCQM-P44 (1&2) and CCQM KC-61). The goal of this CCQM 113 pilot study was to demonstrate the ability to quantify DNA sequences present in a biological tissue using an independent calibration system. The quantification has been performed by QRT-PCR. One laboratory has performed an absolute quantification of both targets by digital PCR without the use of an external standard on sample 1 and found precisely the expected GM concentration. This result reveals the potential of such new technology for absolute quantification of DNA targets. The samples 1 and 3 had a very low number of 1507 targets which has created some difficulties for several laboratories as the unknown samples were too diluted and falling outside calibration curves. The data for the undiluted samples have nevertheless been reported here. Most participating NMIs managed to quantify the 4 samples with high accuracy and reported measurements very close to the assigned value. They could clearly demonstrate their ability to quantify DNA sequences in a biological tissue.
JRC Directorate:Health, Consumers and Reference Materials

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