Established in 2008 as an emerging core facility, the Functional Proteomics Reverse Phase Protein Array (RPPA) Core provides cancer center members with access to a powerful, high-throughput, quantitative cost effective antibody-based assay to characterize basal or ligand-induced protein expression and modification, and time-resolved responses appropriate for systems biology analysis. The RPPA provides information to integrate the consequence of genetic aberrations in cancer, to validate therapeutic targets and to evaluate drug pharmacodynamics. MD Anderson provides 640 square feet of space, additional salary support, and administrative support through the Department of Systems Biology. Services include providing standard operating procedures to investigators for extraction of protein from cells or tumor tissue, processing protein extracts received from investigators, robotic arraying on nitrocellulose-coated slides, and probing with antibodies validated by the RPPA core. Major equipment purchased in part with institutional funding of $320,000 includes a Tecan robotic liquid handling system for serial dilution of cell/tumor lysates and sample transfer, two Aushon 2470 arrayers for printing of cellular/tumor lysates onto nitrocellulose-coated slides and two Dako Universal Staining systems for probing slides with antibodies. Data are analyzed using customized software (MicroVigene and Supercurve Fitting) to determine signal intensity, curve construction, and relative protein concentration. Importantly, the facility validates antibodies to expand the available antibody repertoire. The facility established qulity control processes to improve the quality and accuracy of the RPPA data sets. Since 2008, the facility has processed 47,306 samples from 179 users with a panel of 150 to 180 antibodies. Center members with peer-reviewed funding account for 78% of users, and 20% of total costs are requested from the CCSG. Service utilization grew by an average 14% per year over the past 4 years and 40% over the last 4 years. Publications cited using the RPPA have appeared in high impact journals such as Nature Medicine, Nature Genetics, PNAS and Cancer Cell, etc. In the future, the RPPA Core plans to 1) expand the validated antibody repertoire;2) optimize assay conditions for laser-aided micro-dissected or paraffin-embedded tissue samples;3) explore RPPA applications for body fluids;4) provide forward phase protein array;and 5) develop technology for kinase affinity capture.
Many results from the RPPA Core have been or will be reported in publications and grant applications. To date, 81 publications, including high-profile peer- reviewed journals such as Nature Medicine, Nature Genetics, PNAS, and Cancer Cell, have used data from the Core. In 2011, in recognition of its excellence, the Functional Proteomics RPPA Core was contracted to perform RPPA analysis on all Cancer Genome Atlas samples.
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