title: A well-based reverse-phase protein array of formalin-fixed paraffin-embedded tissue.
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 mm(3) 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.
authors.affiliation: Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, MSC 1500, Bethesda, Maryland, MD, 20892, USA.
authors.forename: Joon-Yong
authors.initials: JY
authors.lastname: Chung
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Hewitt
mesh_terms: D005557:Formaldehyde / Q000378:metabolism*; D006801:Humans; D008297:Male; D016612:Paraffin Embedding; D011467:Prostate / Q000166:cytology; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D016707:Tissue Fixation
chemical_list: D005557:Formaldehyde