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dc.contributor.authorPage, Ken_US
dc.contributor.authorGuttery, DSen_US
dc.contributor.authorZahra, Nen_US
dc.contributor.authorPrimrose, Len_US
dc.contributor.authorElshaw, SRen_US
dc.contributor.authorPringle, JHen_US
dc.contributor.authorBlighe, Ken_US
dc.contributor.authorMarchese, SDen_US
dc.contributor.authorHills, Aen_US
dc.contributor.authorWoodley, Len_US
dc.contributor.authorStebbing, Jen_US
dc.contributor.authorCoombes, RCen_US
dc.contributor.authorShaw, JAen_US
dc.date.accessioned2016-02-19T14:37:51Z
dc.date.available2013-09-06en_US
dc.date.issued2013en_US
dc.date.submitted2016-02-13T22:50:30.111Z
dc.identifier.urihttp://qmro.qmul.ac.uk/xmlui/handle/123456789/11176
dc.description.abstractCirculating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp(®) DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.en_US
dc.format.extente77963 - ?en_US
dc.languageengen_US
dc.language.isoenen_US
dc.relation.ispartofPLoS Oneen_US
dc.rightsCC-BY
dc.subjectBlood Specimen Collectionen_US
dc.subjectBreast Neoplasmsen_US
dc.subjectDNA, Neoplasmen_US
dc.subjectFemaleen_US
dc.subjectHigh-Throughput Nucleotide Sequencingen_US
dc.subjectHumansen_US
dc.subjectMicroRNAsen_US
dc.subjectReagent Kits, Diagnosticen_US
dc.subjectReproducibility of Resultsen_US
dc.titleInfluence of plasma processing on recovery and analysis of circulating nucleic acids.en_US
dc.typeArticle
dc.rights.holder© 2013 Page et al.
dc.identifier.doi10.1371/journal.pone.0077963en_US
pubs.author-urlhttps://www.ncbi.nlm.nih.gov/pubmed/24205045en_US
pubs.issue10en_US
pubs.notesNot knownen_US
pubs.publication-statusPublished onlineen_US
pubs.volume8en_US
dcterms.dateAccepted2013-09-06en_US


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