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

Proteome Science

Methodology

Zhenlin Ju¹  Wenbin Liu¹  Eric Jonasch²  Dimitra Tsavachidou²  Bryan T Hennessy³  Pheroze Tamboli⁴  Huifang Guo⁵  Yiling Lu⁵  Gordon B Mills⁵ 

[1] Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA;Department of Genitourinary Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA;Department of Medical Oncology, Beaumont Hospital, Royal College of Surgeons of Ireland, Dublin, Ireland;Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA;Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA;

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

DOI  :  10.1186/1477-5956-10-56

 received in 2012-02-17, accepted in 2012-09-10,  发布年份 2012

来源: Springer

【 摘 要 】

IntroductionProtein 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.ResultsFresh 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.ConclusionsWith 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.

【 授权许可】

Unknown   
© Guo et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.