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Learn from the Experts
Listen and watch as a distinguished panel of scientific innovators and recognized thought leaders present their research using the RPA platform in a variety of applications.


RPA Overview

RPA Application

Reverse-Phase Protein Lysate Microarrays

Reverse-phase Protein Lysate microarrays (RPAs) provide an exciting new platform for measuring protein expression levels in a large number of biological samples simultaneously. RPA’s, originally introduced by Dr. Lance Liotta of the National Institutes of Health and Dr. Emanuel Petricoin of the FDA, are essentially large arrays of micro dot-blots. They can provide accurate, sensitive and quantitative protein expression data on many samples in a single experiment. Proteomic profiling using RPAs yields more direct answers to functional and pharmacological questions than transcriptional profiling. Dr. Liotta and Dr. Petrcoin are presently co-Directors of the Center for Applied Proteomics and Molecular Medicine at George Mason University.

RPAs were first described by Paweletz et al. to measure subtle quantitative changes in multiple classes of proteins from individual cell types within tissues. Since the initial publication, this technology has also been applied to other biological materials.

Today, there are several notable uses for RPAs such as protein monitoring for bio-marker discovery and the monitoring of signal transduction proteins in response to various biological stimuli. For example, government researchers and pharmaceutical companies are using RPAs to map protein signaling pathways, assess drug target and biomarker expression, and understand a drug candidate’s mechanism of action. Dr. Nishizuka (Nishizuka et al.) of the National Institutes of Health has created 10,000-element lysate arrays for pathway mapping, thereby permitting detailed time-course studies to be performed of protein response to cell stimulation. The ability to quantitatively track, for example, the phosphorylation cascade down one branch of a pathway versus another can guide researchers in developing compounds that best elicit the desired therapeutic effects.

Clinical researchers are also examining RPAs as a potential early means of implementing personalized cancer treatment (Espina et al.). By examining the expression levels of key proteins in cancer cells isolated via laser-capture microdissection, researchers can identify the exact nature of protein mis-expression. The goal of this work is to allow clinicians to recommend therapy tuned to the exact nature of a patient’s cancer.

One of the most sensitive detection methodologies for RPAs involves colorimetric detection via amplification steps involving tagged secondary antibodies. The RPA data, viewed as series of dilution curves, is a sensitive, quantitative, and much higher throughput alternative to Western blotting. Aushon BioSystems’ 2470 arrayer is a proven technology for building exceptionally high quality Reverse-phase Protein Lysate microarrays (RPA). The 2470 can produce arrays of the often viscous lysate material with the linearity and consistency to permit data extraction from dilution series curves.

Related Links:
Molecular Translational Technologies MTP/CCR/NCI/NIH
GMU Center for Applied Proteomics and Molecular Medicine
Grace Bio-Labs, Inc.
SCHOTT Technical Glass Solutions GmbH

References:
Paweletz et al. 2001. Oncogene 20: 1981-1989
Espina et al. 2003. Proteomics 3: 2091-2100
Nishizuka et al. 2003a. PNAS 100: 14229-14234
Nishizuka et al. 2003b. Cancer Research 63: 5243-5250
Nishizuka et al. 2006. BioTechniques 40(4): 442-448
Nishizuka et al., 2006, European Journal of Cancer
Wulfkuhle et al., 2006, Vol 3 No 5 Nature Clinical Practice Oncology
Petricoin et al., 2007, Cancer Res; 67: (7)
Shankavaram et al., 2007, Molecular Cancer Therapeutics: 6(3) 820-832

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