Multiple gene expression analyses in paraffin-embedded tissues by TaqMan low-density array: Application to hedgehog and Wnt pathway analysis in ovarian endometrioid adenocarcinoma.

Author(s): Steg A, Wang W, Blanquicett C, Grunda JM, Eltoum IA, Wang K, Buchsbaum DJ, Vickers SM, Russo S, Diasio RB, Frost AR, LoBuglio AF, Grizzle WE, Johnson MR

Publication: J Mol Diagn, 2006, Vol. 8, Page 76-83

PubMed ID: 16436637 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the performance of RNA isolated from case-matched fresh frozen and formalin-fixed, paraffin embedded (FFPE) tissues on TaqMan low-density arrays.

   Summary of Findings:

   TaqMan low density array yielded expression data for the 48 genes evaluated for all specimens regardless of the method of preservation. PCR amplification efficiencies were comparable among snap frozen and FFPE specimens. Relative expression of the 48 genes examined (to the RPLP0 gene) were very strongly correlated among snap frozen and FFPE specimens (R=0.92). Of the genes evaluated 9% were differentially expressed among frozen and FFPE specimens. Individual analysis of a subset of genes by TaqMan real-time qRT-PCR confirmed results obtained with the low density array, illustrating a very strong correlation in gene expression among snap frozen and FFPE specimens.

   Biospecimens

   • Tissue - Ovary

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen

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2. Study Purpose

   The purpose of this study was to determine if RNA extracted from snap frozen and FFPE specimens can be successfully amplified prior to analysis by TaqMan low density array. Four different commercially available kits were evaluated.

   Summary of Findings:

   RNA amplification was unsuccessful for all FFPE specimens regardless of the kit used. While RNA amplification of snap frozen specimens did not alter targeted gene expression levels, expression profiles were affected, with as many as 57% of the genes examined undetectable after RNA amplification. The Full Spectrum amplification system, which utilizes random hexamer primers, exhibited the highest correlation among unamplified and amplified gene expression profiles, with 29% of the genes examined undetectable after RNA amplification.

   Biospecimens

   • Tissue - Ovary

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen
   Real-time qRT-PCR Specific	Nucleic acid amplification	Full Spectrum RNA amplification kit No amplification Ovation RNA amplification system MessageAmp aRNA kit RiboAmp RNA amplification kit

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