Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.
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.
Linton
Kim
K
Cancer Research UK Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK. kim.linton@christie.nhs.uk
Hey
Yvonne
Y
Dibben
Sian
S
Miller
Crispin
C
Freemont
Anthony
A
Radford
John
J
Pepper
Stuart
S
D002277:Carcinoma / Q000235:genetics / Q000473:pathology*; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D005615:Freezing; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016612:Paraffin Embedding / Q000379:methods; D012313:RNA / Q000032:analysis; D012680:Sensitivity and Specificity; D046888:Tissue Array Analysis / Q000379:methods*; D014015:Tissue Banks; D016707:Tissue Fixation / Q000379:methods; D014158:Transcription, Genetic; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000473:pathology*
D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D005404:Fixatives; D005557:Formaldehyde; D012313:RNA