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A Well-Based Reverse-Phase Protein Array of Formalin-Fixed Paraffin-Embedded Tissue

  • Joon-Yong Chung
  • Stephen M. Hewitt
Part of the Methods in Molecular Biology book series (MIMB, volume 1312)

Abstract

Biomarkers from tissue-based proteomic studies directly contribute to defining disease states as well as promise to improve early detection or provide for further targeted therapeutics. In the clinical setting, tissue samples are preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks for histological examination. However, proteomic analysis of FFPE tissue is complicated due to the high level of covalently cross-linked proteins arising from formalin fixation. To address these challenges, we developed well-based reverse-phase protein array (RPPA). This approach is a robust protein isolation methodology (29.44 ± 7.8 μg per 1 mm3 of FFPE tissue) paired with a novel on electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots, demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, dried up and vacuum-sealed, remain stable up to 2 months at room temperature. This methodology is directly applicable to FFPE tissue and presents the direct opportunity of addressing hypothesis within clinical trials and well-annotated clinical tissue repositories.

Key words

Formalin-fixed Paraffin-embedded Tissue lysate Protein extraction Proteomics Reverse-phase protein array Electrochemiluminescence 

Notes

Acknowledgments

We thank Kris Ylaya for technical assistance. This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Laboratory of Pathology, Center for Cancer ResearchNational Cancer Institute, National Institutes of HealthMarylandUSA

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