Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting.

Author(s): Nirmalan NJ, Harnden P, Selby PJ, Banks RE

Publication: J Pathol, 2009, Vol. 217, Page 497-506

PubMed ID: 19156775 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of preservation method, extraction buffer, and HIAR regime on the extraction of full-length proteins from normal and renal cell carcinoma specimens. Specimens were stored for less than 2 years prior to this study.

   Summary of Findings:

   The optimum extraction protocol for obtaining full length proteins from FFPE renal tissue included using Laemmli buffer, rather than QProteome buffer, modified RIPA buffer, pH 7.6, or ACN-ammonium bicarbonate. HIAR at 105 degrees C for 20 min was superior to 10 min or no HIAR in terms of protein yields. HIAR at 80 degrees C for an additional 20 min also yielded good, but somewhat more variable results. 63% of the protein was extracted from FFPE tissue compared to frozen tissue. FFPE protein extracts exhibited considerably more background staining than extracts from frozen tissue, and the background staining of FFPE protein extracts was not improved following acetone precipitation. SELDI-TOF profiles were similar but not identical between frozen and FFPE extracts. While some variation was observed between frozen and FFPE specimens in fold-changes in the expression of 7 biomarkers between normal and renal cell carcinoma tissue, the direction of change was generally preserved, and there were significant correlations between fresh and FFPE tissues (r=0.7292). Western blot analysis of FFPE protein extracts revealed variable detection of membrane proteins.

   Biospecimens

   • Tissue - Kidney

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	1D/2D gels
   Protein	Western blot
   Protein	Spectrophotometry
   Protein	SELDI-TOF MS
   Protein	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen
   Western blot Specific	Targeted peptide/protein	Beta actin Carbonic anhydrase IX GAPDH Thymidine phosphorylase Ubiquinol-cytochrome c reductase core protein-1 Ubiquinol-cytochrome c reductase core protein-2 Heat shock protein 60 Heat shock protein 70 Beta tubulin Fascin Beta dystroglycan Golgin-84 NaK-ATPase
   Analyte Extraction and Purification	Analyte isolation method	Laemmli extraction buffer QProteome FFPE Tissue Kit Modified RIPA buffer, pH 7.6 ACN-ammonium bicarbonate
   Analyte Extraction and Purification	Temperature of heat-induced retrieval	No boiling 105 degrees C for 10 min 105 degrees C for 20 min 105 degrees C for 20 min, 80 degrees C for 20 min 100 degrees C for 20 min, 80 degrees C for 2 h 100 degrees C for 5 min
   Analyte Extraction and Purification	Analyte purification	Acetone precipitation No acetone precipitation

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