Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.

2.50
Hdl Handle:
http://hdl.handle.net/10541/87535
Title:
Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.
Authors:
Linton, Kim M ( 0000-0002-3294-1548 ) ; Hey, Yvonne; Dibben, Sian; Miller, Crispin J; Freemont, Anthony J; Radford, John A ( 0000-0001-7898-2786 ) ; Pepper, Stuart D
Abstract:
Microarray 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.
Affiliation:
Cancer Research UK Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK. kim.linton@christie.nhs.uk
Citation:
Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays. 2009, 47 (1):587-96 BioTechniques
Journal:
BioTechniques
Issue Date:
Jul-2009
URI:
http://hdl.handle.net/10541/87535
DOI:
10.2144/000113169
PubMed ID:
19594443
Type:
Article
Language:
en
ISSN:
1940-9818
Appears in Collections:
Applied Computational Biology and Bioinformatics; All Paterson Institute for Cancer Research; Medical Oncology; Molecular Biology Core Facility

Full metadata record

DC FieldValue Language
dc.contributor.authorLinton, Kim Men
dc.contributor.authorHey, Yvonneen
dc.contributor.authorDibben, Sianen
dc.contributor.authorMiller, Crispin Jen
dc.contributor.authorFreemont, Anthony Jen
dc.contributor.authorRadford, John Aen
dc.contributor.authorPepper, Stuart Den
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.en
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
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