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
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.
Analyte Technology Platform RNA Real-time qRT-PCR Protein Immunohistochemistry DNA FISH
Classification Pre-analytical Factor Value(s) Real-time qRT-PCR Specific Targeted nucleic acid p21/WAF/Cip1
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
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.
- Neoplastic - Carcinoma
- Neoplastic - Benign
Analyte Technology Platform RNA RT-PCR
Classification Pre-analytical Factor Value(s) RT-PCR Specific Targeted nucleic acid EGFR
Biospecimen Aliquots and Components Biospecimen heterogeneity Biospecimen core