Here, we describe the use of reverse phase protein arrays rppas or rplas to profile signaling proteins in 56 breast cancers and matched normal tissue. Apr 17, 20 reverse phase protein array or rppa is a method to quantitate protein levels in lysates from cells or tissues. Functional proteomics by reverse phase protein array array on nitrocellulose coated slides probe with validated antibodies, signal detection by dab colorimetric reaction serial dilution 2. Oct 14, 2011 video showing nanoprint microarrayer manufacturing protein lysate microarrays onto grace biolabs nitrocellulose slides.
Overton 4, lel eory 4, marie odonnell 1,5, dana faratian 6, thomas powles 7, david j. B incubation with a highly, targetspecific primary antibody to detect. The reverse phase protein array rppa assay platform has become a powerful tool for protein expression profiling, biomarker discovery, and cancer diagnostics. Most of them either function on the basis of forward phase protein arrays or reverse phase protein arrays 11.
From experimental design towards targeted biomarker discovery astrid wachter 1, stephan bernhardt 2, tim beissbarth 1 and ulrike korf 2, 1 statistical bioinformatics, department of medical statistics, university medical center goettingen, humboldtallee 32, d37073 goettingen, germany. Oct 31, 2014 reverse phase protein array rppa technology evolved from the advent of miniaturized immunoassays and gene microarray technology. The reversephase protein array rppa assay platform has become a powerful tool for protein expression profiling, biomarker discovery, and cancer diagnostics. Furthermore, we provide centralized, standardized and qualitycontrolled services to investigators not only throughout md anderson, but around the world, as well as. As the demand for patient tailored therapies increases so. Reverse phase chromatography is commonly coupled with mass spectrometry in an effort to quantify the protein that is eluted from the column. Implementing reverse phase protein array profiling as a. Reverse phase protein array or rppa is a method to quantitate protein levels in lysates from cells or tissues. Reverse phase protein microarrays which capture disease. In contrast to previous protein arrays that immobilize the probe, our reverse phase protein array immobilizes the whole repertoire of patient proteins that represent the state of individual.
In essence, the rppa technique for molecular profiling is based on a miniaturized dot blot in a microarray format. It extends the power of immunoblotting to provide a quantitative analysis of the differential expression of active usually phosphorylated or cleaved and parental proteins. The lysate is arrayed onto the microarray and probed with antibodies against the target protein of interest. Proteomicsprotein separations chromatographyreversed phase. Profiling b cell chronic lymphocytic leukemia by reverse. Protein arrays are described for screening of molecular markers and pathway targets in patient matched human tissue during disease progression. We analyzed b lymphocytes from 57 patients with cll and 11 healthy subjects. Pdf spatial normalization of reverse phase protein array.
Reverse phase protein array rppa is a new highthroughput technology whereby a multiplexed assessment of protein expression on more than 100 differing proteins can be carried out simultaneously on up to tumors samples. Spatial normalization of reverse phase protein array data. The introduction of gene expression microarray technology in 1995 schena et al. In the case of forward phase protein arrays numerous capture antibodies are printed on a solid phase and are exposed to a single protein. Omahony, jyoti nanda, alexander laird, peter mullen, helen caldwell, ian m. Variable slope normalization of reverse phase protein arrays. The reverse phase protein array rppa core provides investigators with a powerful, high. We propose to use a reverse phase protein array rppa approach to investigate new potential biomarkers of oocyte competence in human ccs at the protein level, an approach that is already used in cancer research to identify biomarkers in clinical diagnostics. Quantification of biomarker proteins using reverse. Rppa is highly sensitive and requires only very small protein samples. Policies and guidelines for sample preparation 5 2014. Rppanalyzer analyze reverse phase protein array data users guide.
One reverse phase approach that uses lysed homogenized samples is the protein lysate or reverse phase protein array rppa. Preclinical evaluation and reverse phase protein arraybased. The difference between the two refers to the way the sample is applied. Functional proteomics by reverse phase protein array. On forwardphase protein arrays, the sample is incubated on the array so that. Reverse phase protein arrays represent an innovative technology that allows studying protein expression levels and the activation status of cell signaling pathways. Reverse phase protein arrays rppa allow for the specific detection and quantification of proteins in a reproducible and highly parallelized manner paweletz et al. Different pathways were assessed for activation expression of key signaling proteins. This book provides practical guidance on all aspects of reverse phase protein array rppa technology. Rppanalyzer is a statistical tool developed to read reverse phase protein array data, to perform the basic data analysis and to visualize the resulting biological information. Reverse phase protein array rppa technology is a powerful technique for measuring the activities of proteins from tissue and cellderived lysate. The use of reverse phase protein arrays rppa to explore protein expression variation within individual renal cell cancers fiach c. Although primary efforts with this method have focused on cancer tissue profiling, this method has general utility for monitoring protein expression in experimental systems biology.
The complexity of rppa data also suggests implementing a data management system for standardization of rppa data. Rppa can be used as a tool to explore cellular signaling pathway activation that relates to the biology of cancer. Reverse phase protein arrays provide either a low throughput or high throughput methodology for quantifying proteins and their posttranslationally modified forms in both cellular and noncellular samples. Reverse phase protein array rppa technology is a highthroughput antibody and microarraybased approach for the rapid profiling of levels of proteins and protein posttranslational modifications. The wikipedia information on this type of microarray is pretty good. To assure rppa assay generates the highest quality data requires samples to be prepared according to specific protocols and policies and to meet the specifications as outlined below. Evaluation of reverse phase protein array rppabased pathwayactivation profiling in 84 nonsmall cell lung cancer nsclc cell lines as platform for cancer proteomics and biomarker discovery. It extends the power of immunoblotting to provide a quantitative analysis of the differential expression of active. This book provides practical guidance on all aspects of reverse phase protein array rppa technology, which permits the quantification of protein levels in cell or tissue lysates. The three major classes of protein microarray are functional arrays, forward phase or antibody arrays, and lysate or reverse phase protein arrays rppas. The use of reverse phase protein arrays rppa to explore. Discover our platform for reverse phase protein array. Profiling phosphosignaling networks in breast cancer. Stages in reversed phase chromatography edit the stages in reversed phase chromatography are very similar to the stages found in ion exchange chromatography.
Reverse phase protein microarrays are a direct descendent of miniaturized immunoassays ekins, 1989, ekins and chu, 1991. The role of rppa data in integrative analyses is discussed, and recent results are presented on the various applications of rppa, for example for drug discovery and evaluation of drug efficacy. In reverse phase arrays, numerous protein samples are printed in discrete spots on the array, which is then probed with a single validated antibody, simultaneously measuring the level of a single protein in multiple samples. In addition, the latest results are presented from laboratories across the world where experts are successfully running the challenging rppa data platform. Reverse phase protein arrays from technical and analytical. In the case of forward phase protein arrays numerous capture antibodies are printed on a solid phase and are exposed to a single protein lysate figure 1, allowing detection of multiple proteins in a single sample. May 15, 2010 reverse phase protein array rppa is a new highthroughput technology whereby a multiplexed assessment of protein expression on more than 100 differing proteins can be carried out simultaneously on up to tumors samples. Reverse phase protein arraysquantitative assessment of. Overton, lel eory, marie odonnell, dana faratian, thomas powles, david j. Reverse phase protein microarrays advance to use in clinical.
Array technologies including dna microarrays, qpcr arrays, antibody arrays, recombinant protein arrays, tissue arrays, and reverse phase protein arrays rppa are among the methods being used for this task. Semiquantitative measurement of specific proteins in human. Cells are isolated from various tissues of interest and are lysed. Reverse phase protein microarray rppa involve complex samples, such as tissue lysates. This video of the becker lab from the technische universitat munchen describes in. The rpackage provides different functions to compare protein expression levels of different samples and to normalize the data. Many of the platforms in use are complementary to each other, but only a few. Pdf evaluation of reverse phase protein array rppabased.
Omahony 1, jyoti nanda 1, alexander laird 1, peter mullen 2, helen caldwell 3, ian m. Profiling phosphosignaling networks in breast cancer using. Reverse phase protein arrays rppa experimentation involves a printing of samples in a neatly organized array format onto, for example, nitrocellulosecoated glass slides. Evaluation of reverse phase protein array rppa based pathwayactivation profiling in 84 nonsmall cell lung cancer nsclc, cell lines as platform for cancer proteomics and biomarker discovery. Pdf evaluation of reverse phase protein array rppa. The zeptosens rppa platform is employed for protein expression analysis. Video showing nanoprint microarrayer manufacturing protein lysate microarrays onto grace biolabs nitrocellulose slides. Rppanalyzer is a statistical tool developed to read reversephase protein array data, to perform the basic data analysis and to visualize the resulting biological information. Protein extraction and reverse phase protein array rppa rppa is a high throughput antibody based technique used for functional proteomic assessments of a large number of tumour samples and it offers a platform for comparison of the relative protein expression between these samples. Reverse phase protein microarrays which capture disease progression show activation of prosurvival pathways at the cancer invasion front skip to main content thank you for visiting. Functional arrays involve immobilization of peptides or whole proteins and are used for applications such as discovery of.