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dc.contributor.authorLinton, Kim M
dc.contributor.authorHey, Yvonne
dc.contributor.authorDibben, Sian
dc.contributor.authorMiller, Crispin J
dc.contributor.authorFreemont, Anthony J
dc.contributor.authorRadford, John A
dc.contributor.authorPepper, Stuart D
dc.date.accessioned2009-12-08T12:20:55Z
dc.date.available2009-12-08T12:20:55Z
dc.date.issued2009-07
dc.identifier.citationMethods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays. 2009, 47 (1):587-96 BioTechniquesen
dc.identifier.issn1940-9818
dc.identifier.pmid19594443
dc.identifier.doi10.2144/000113169
dc.identifier.urihttp://hdl.handle.net/10541/87535
dc.description.abstractMicroarray gene expression profiling of formalin-fixed paraffin-embedded (FFPE) tissues is a new and evolving technique. This report compares transcript detection rates on Affymetrix U133 Plus 2.0 and Human Exon 1.0 ST GeneChips across several RNA extraction and target labeling protocols, using routinely collected archival FFPE samples. All RNA extraction protocols tested (Ambion-Optimum, Ambion-RecoverAll, and Qiagen-RNeasy FFPE) provided extracts suitable for microarray hybridization. Compared with Affymetrix One-Cycle labeled extracts, NuGEN system protocols utilizing oligo(dT) and random hexamer primers, and cDNA target preparations instead of cRNA, achieved percent present rates up to 55% on Plus 2.0 arrays. Based on two paired-sample analyses, at 90% specificity this equalled an average 30 percentage-point increase (from 50% to 80%) in FFPE transcript sensitivity relative to fresh frozen tissues, which we have assumed to have 100% sensitivity and specificity. The high content of Exon arrays, with multiple probe sets per exon, improved FFPE sensitivity to 92% at 96% specificity, corresponding to an absolute increase of ~600 genes over Plus 2.0 arrays. While larger series are needed to confirm high correspondence between fresh-frozen and FFPE expression patterns, these data suggest that both Plus 2.0 and Exon arrays are suitable platforms for FFPE microarray expression analyses.
dc.language.isoenen
dc.subjectUterine Cervical Canceren
dc.subject.meshCarcinoma
dc.subject.meshDNA Primers
dc.subject.meshDNA Probes
dc.subject.meshDNA, Complementary
dc.subject.meshExons
dc.subject.meshFemale
dc.subject.meshFixatives
dc.subject.meshFormaldehyde
dc.subject.meshFreezing
dc.subject.meshGene Expression
dc.subject.meshGene Expression Profiling
dc.subject.meshHumans
dc.subject.meshOligonucleotide Array Sequence Analysis
dc.subject.meshParaffin Embedding
dc.subject.meshRNA
dc.subject.meshSensitivity and Specificity
dc.subject.meshTissue Array Analysis
dc.subject.meshTissue Banks
dc.subject.meshTissue Fixation
dc.subject.meshTranscription, Genetic
dc.subject.meshUterine Cervical Neoplasms
dc.titleMethods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.en
dc.typeArticleen
dc.contributor.departmentCancer Research UK Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK. kim.linton@christie.nhs.uken
dc.identifier.journalBioTechniquesen
html.description.abstractMicroarray gene expression profiling of formalin-fixed paraffin-embedded (FFPE) tissues is a new and evolving technique. This report compares transcript detection rates on Affymetrix U133 Plus 2.0 and Human Exon 1.0 ST GeneChips across several RNA extraction and target labeling protocols, using routinely collected archival FFPE samples. All RNA extraction protocols tested (Ambion-Optimum, Ambion-RecoverAll, and Qiagen-RNeasy FFPE) provided extracts suitable for microarray hybridization. Compared with Affymetrix One-Cycle labeled extracts, NuGEN system protocols utilizing oligo(dT) and random hexamer primers, and cDNA target preparations instead of cRNA, achieved percent present rates up to 55% on Plus 2.0 arrays. Based on two paired-sample analyses, at 90% specificity this equalled an average 30 percentage-point increase (from 50% to 80%) in FFPE transcript sensitivity relative to fresh frozen tissues, which we have assumed to have 100% sensitivity and specificity. The high content of Exon arrays, with multiple probe sets per exon, improved FFPE sensitivity to 92% at 96% specificity, corresponding to an absolute increase of ~600 genes over Plus 2.0 arrays. While larger series are needed to confirm high correspondence between fresh-frozen and FFPE expression patterns, these data suggest that both Plus 2.0 and Exon arrays are suitable platforms for FFPE microarray expression analyses.


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