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dc.contributor.authorThormar, Hans G
dc.contributor.authorGudmundsson, Bjarki
dc.contributor.authorEiriksdottir, Freyja
dc.contributor.authorKil, Siyoen
dc.contributor.authorGunnarsson, Gudmundur H
dc.contributor.authorMagnusson, Magnus Karl
dc.contributor.authorHsu, Jason C
dc.contributor.authorJonsson, Jon J
dc.date.accessioned2014-08-08T13:44:43Z
dc.date.available2014-08-08T13:44:43Z
dc.date.issued2013-04
dc.date.submitted2013
dc.identifier.citationClin. Chem. 2013, 59 (4):667-74en
dc.identifier.issn1530-8561
dc.identifier.pmid23378568
dc.identifier.doi10.1373/clinchem.2012.193839
dc.identifier.urihttp://hdl.handle.net/2336/324522
dc.descriptionTo access publisher's full text version of this article click on the hyperlink at the bottom of the pageen
dc.description.abstractThe causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids.
dc.description.abstractWe used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis.
dc.description.abstractThe relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples.
dc.description.abstractVariation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis.
dc.description.sponsorshipTechnical Development Fund at Rannis/110473-0612 Icelandic Center for Research Funds University of Iceland Research Fund Landspitali Research Funden
dc.language.isoenen
dc.publisherAmer Assoc Clinical Chemistryen
dc.relation.urlhttp://dx.doi.org/10.1373/clinchem.2012.193839en
dc.relation.urlhttp://www.clinchem.org/content/59/4/667.full.pdfen
dc.rightsArchived with thanks to Clinical chemistryen
dc.subjectDNA kjarnsýraen
dc.subject.meshDNAen
dc.subject.meshDNA, Complementaryen
dc.subject.meshElectrophoresis, Gel, Two-Dimensionalen
dc.subject.meshOligonucleotide Array Sequence Analysisen
dc.subject.meshReproducibility of Resultsen
dc.titleImportance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.en
dc.typeArticleen
dc.contributor.departmentUniv Iceland, Dept Biochem & Mol Biol, IS-101 Reykjavik, Iceland, Lifeind Ehf, Reykjavik, Iceland, Ohio State Univ, Dept Stat, Columbus, OH 43210 USA, Univ Iceland, Dept Pharmacol & Toxicol, IS-101 Reykjavik, Iceland, Landspitali Natl Univ Hosp, Dept Genet & Mol Med, Reykjavik, Icelanden
dc.identifier.journalClinical chemistryen
dc.rights.accessLandspitali Access - LSH-aðganguren
html.description.abstractThe causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids.
html.description.abstractWe used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis.
html.description.abstractThe relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples.
html.description.abstractVariation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis.


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