Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5' nuclease quantitative reverse transcription-polymerase chain reaction.

Author(s): Godfrey TE, Kim SH, Chavira M, Ruff DW, Warren RS, Gray JW, Jensen RH

Publication: J Mol Diagn, 2000, Vol. 2, Page 84-91

PubMed ID: 11272893 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to optimize RNA extraction and reverse transcription for archival FFPE specimens (fixed for 18 hours).

   Summary of Findings:

   The authors report that a 3-day proteinase K incubation at 55 degrees C yielded a greater quantity of RNA with longer fragment lengths than a 1-day proteinase K incubation. RNA yield was independent of paraffin block storage duration. Low OD 260/280 ratios were improved after two sequential extractions with Trizol, thereby removing potential impurities and genomic contamination, and improving RT consistency. The authors note that the efficiency of real-time qRT-PCR analysis of archival FFPE specimens was improved by doubling the concentration of enzyme, nucleotides, hexamers, and MgCl2 in the final reaction volume, while linear amplification was maintained.

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protein digestion	3 d Proteinase K incubation, 55 degrees C 1 d Proteinase K incubation, 55 degrees C
   Analyte Extraction and Purification	Analyte isolation method	2 Sequential Trizol extractions 1 Trizol extraction
   Storage	Storage duration	7 yr 10 yr 15 yr

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2. Study Purpose

   The purpose of this study was to evaluate whether amplicon length influences real-time qRT-PCR quantitation of RNA from FFPE tissues.

   Summary of Findings:

   Real-time qRT-PCR analysis of short fragments (<131 bp) resulted in the smallest difference in raw Ct values between RNA extracted from FFPE and freshly procured specimens.

   Biospecimens

   • Tissue - Liver

   Preservative Types

   • Formalin
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Length of gene fragment	99 bp 115 bp 131 bp 175 bp 205 bp 291 bp
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh)

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3. Study Purpose

   The purpose of this study was to evaluate whether a fixation delay at room temperature in phosphate-buffered saline for up to 12 hours influenced mRNA quantitation by real-time qRT-PCR.

   Summary of Findings:

   The authors report that delaying tissue fixation for up to 12 hours did not significantly influence relative expression of six mRNA species: GAPDH, VEGF, KDR, FLT, Her-2, and ZNF217. Of note, real-time qRT-PCR data collected from FFPE specimens was relative to beta-Gus, and normalized to fresh (non-fixed) specimens. Gene-specific fixation effects were observed, although in general raw Ct values averaged 5 cycles higher in FFPE specimens compared to freshly procured specimens.

   Biospecimens

   • Tissue - Liver

   Preservative Types

   • Formalin
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	1 h 2 h 4 h 8 h 12 h
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh)
   Real-time qRT-PCR Specific	Targeted nucleic acid	GAPDH VEGF KDR FLT Her-2 ZN217

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