Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.

Author(s): Specht Katja, Richter Thomas, Muller Ulrike, Walch Axel, Werner Martin, Hofler Heinz

Publication: Am J Pathol, 2001, Vol. 158, Page 419

PubMed ID: 11159180 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the optimal microscale RNA extraction method for microdissected FFPE archived tissue, and to confirm that quantities are sufficient for real-timer quantitative RT-PCR analysis.

   Summary of Findings:

   The microscale RNA extraction method that resulted in the greatest RNA yield and the most reliable qRT-PCR results for p21/WAF1/Cip1 included proteinase K digestion at 60 degrees C followed by organic extraction. Sufficient mRNA was extracted from FFPE microdissected tumors for successful real-time qRT-PCR analysis of HER-2/neu gene expression; FISH and immunohistochemistry of full tissue sections confirmed real-time qRT-PCR results.

   Biospecimens

   • Tissue - Liver

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	p21/WAF/Cip1 HER-2/neu
   Analyte Extraction and Purification	Analyte isolation method	Proteinase K digestion at 60 degrees C and organic extraction Proteinase K digestion at 42 degrees C Acidic guanidinium thiocyanate-phenol chloroform Oligo d(T) coupled magnetic beads Guanidinium thiocyanate lysis with silica-matrix spin technology
   Biospecimen Aliquots and Components	Aliquot size/volume	Microdissected cells Full FFPE section

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2. Study Purpose

   The purpose of this study was to determine possible effects on gene expression (EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, HPRT, PGK) of delayed fixation as a function of the rate of fixative penetration in large specimens (>7 cm) that were fixed in formalin for 20 h. Liver and uterus specimens were analyzed.

   Summary of Findings:

   Dissected regions from the periphery to the core of the specimen did not differ in expression for any of the following 5 genes analyzed by RT-PCR: EGFR, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2.

   Biospecimens

   • Tissue - Liver
   • Tissue - Uterus

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   RT-PCR Specific	Targeted nucleic acid	EGFR HER-2/neu FGF-R4 p21/WAF1/Cip1 MDM2
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Biospecimen core Biospecimen periphery

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