Impact of fixative on recovery of mRNA from paraffin-embedded tissue.

Author(s): Benchekroun M, DeGraw J, Gao J, Sun L, von Boguslawsky K, Leminen A, Andersson LC, Heiskala M

Publication: Diagn Mol Pathol, 2004, Vol. 13, Page 116-25

PubMed ID: 15167013 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the effects of storage and fixation of colon and ovarian specimens with Carnoy's or methanol-acetone on RNA recovery and amplification, IHC staining, and morphology.

   Summary of Findings:

   RNA from tissues fixed in Carnoy's was less fragmented than that from specimens fixed in methanol-acetone. B-actin transcript abundance was not significantly different between the two fixatives. Storage of specimens that were fixed in Carnoy's solution for 2 years at room temperature resulted in a non-significant 23% reduction in B-actin transcripts as quantified by amplification of a 248 bp region. However, in laser microdissected specimens, a significant 8-fold reduction was observed in this same fragment when specimens were stored for 2 years, versus 1 year, after fixation in Carnoy's solution. Methanol-acetone and Carnoy's-fixed specimens showed good preservation of IHC staining and morphology.

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Ovary

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR
   Morphology	H-and-E microscopy
   Protein	Immunohistochemistry
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Methanol-acetone Carnoy's solution
   Immunohistochemistry Specific	Targeted peptide/protein	Cytokeratin (CAM5.2) Vimentin CD45 Cyclin A
   RT-PCR Specific	Targeted nucleic acid	Beta-actin
   Storage	Time at room temperature	1 year 2 years

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2. Study Purpose

   The purpose of this study was to compare the effects of preservation of colon and ovarian specimens with Carnoy's solution or snap-freezing on RNA amplification and microarray analysis.

   Summary of Findings:

   Fixation in Carnoy's solution allowed for detection of rare transcripts by RT-PCR. A comparison of one of the rare transcripts, survivin, showed higher levels in 2 Carnoy's solution-fixed specimens than in frozen specimens while lower levels were observed in 2 others. Amplification of RNA using T7 was possible in 5 of 8 Carnoy's-fixed specimens compared with 7 of 8 frozen specimens. Microarray analysis of Carnoy's-fixed and frozen specimens showed a high degree of correlation (R2= 0.94-0.96).

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Ovary

   Preservative Types

   • Frozen
   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	RT-PCR
   RNA	RNA amplification

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Carnoy's solution Snap frozen
   RT-PCR Specific	Targeted nucleic acid	Survivin Human carcinoembryonic antigen Human telomerase reverse transcriptase (hTrT) MAGE-3 PRAME

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