Effect of duration of fixation on quantitative reverse transcription polymerase chain reaction analyses.

Author(s): Macabeo-Ong M, Ginzinger D, Dekker N, McMilan A, Regezi J, Wong D, Jordan R

Publication: Mod Pathol, 2002, Vol. 15, Page 979

PubMed ID: 12218216 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   To determine if the duration of formalin fixation in FFPE tissue impacts mRNA expression of targeted tumor markers (EGFR, p21, MMP-1, VEGF). Specimens collected from 9 patients were examined.

   Summary of Findings:

   Sufficient mRNA for real time qRT-PCR analysis was isolated from all specimens at all fixation times. Formalin fixation for 48 h or more adversely impacted PCR efficiency in a marker dependent manner: MMP-1 and VEG-F mRNA expression increased and p21 expression decreased, while EGFR remained unaltered in samples fixed for 48 h when compared to fresh frozen controls. Results of mRNA expression analysis for all markers in samples fixed for 24 h were similar to fresh frozen controls.

   Biospecimens

   • Tissue - Oral Cavity

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Other
   • Normal

   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
   Biospecimen Preservation	Time in fixative	0 h 24 h 48 h 72 h 1 week
   Real-time qRT-PCR Specific	Targeted nucleic acid	p21 EGFR MMP-1 VEGF

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2. Study Purpose

   The purpose of this study was to determine if mRNA and protein expression remain correlated in archived FFPE oral squamous cell carcinomas specimens in comparison to expression in normal epithelium. Tumor markers (EGFR, p21, MMP-1, VEGF) were examined in 15 FFPE archived specimens that had been fixed in formalin for 24 h.

   Summary of Findings:

   The percentage of archived FFPE oral squamous cell carcinoma specimens that exhibited overexpression compared to normal epithelium were similar for real-time qRT-PCR and immunohistochemistry, respectively, for EGFR (93% versus 67%), MMR-1 (93% with either platform), and VEGF (93% versus 73% of specimens). In contrast, p21 mRNA was not overexpressed among any of the 15 archival FFPE specimens analyzed although 93% of specimens exhibited elevated protein expression.

   Biospecimens

   • Tissue - Oral Cavity

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal
   • Neoplastic - Other

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Immunohistochemistry Specific	Targeted peptide/protein	EGFR MMR-1 VEGF p21
   Real-time qRT-PCR Specific	Targeted nucleic acid	EGFR MMR-1 VEGF p21
   Preaquisition	Diagnosis/ patient condition	Oral squamous cell carcinoma Normal epithelium
   Immunohistochemistry Specific	Technology platform	Immunohistochemistry Real-time qRT-PCR

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