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Open Access Highly Accessed Methodology

An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays

Huifang Guo1, Wenbin Liu2, Zhenlin Ju2, Pheroze Tamboli3, Eric Jonasch4, Gordon B Mills1, Yiling Lu1, Bryan T Hennessy5 and Dimitra Tsavachidou4*

Author Affiliations

1 Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

2 Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

3 Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

4 Department of Genitourinary Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

5 Department of Medical Oncology, Beaumont Hospital, Royal College of Surgeons of Ireland, Dublin, Ireland

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Proteome Science 2012, 10:56  doi:10.1186/1477-5956-10-56

Published: 24 September 2012

Abstract

Introduction

Protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA) is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA.

Results

Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples.

The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p < 0.05) in at least one cell or tissue type, with only 23 markers common in all three sample sets. In addition, FFPE preparations yielded biologically meaningful observations related to pathway signaling status in cell lines, and classification of renal tissues.

Conclusions

With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type.

Keywords:
Formalin-fixed; Paraffin-embedded tissue; Protein extraction; Reverse phase protein array; Breast cancer; Renal cancer