Protein extracts obtained from cultured cells or tissues are serially diluted, spotted onto nitrocellulose-coated microscope slides and probed for a specific protein or protein modification via antibodies that have been validated for RPPA use. Probing for protein modifications of samples enables functional proteomics studies applicable to systems biology analyses. Probed signals are scanned to obtain digital images of the slides; spot densities from the digital images are quantified via software to determine relative levels of the protein for all samples on the slide. Software is also used to determine staining quality of each slide. Each slide is probed with one antibody; replicate slides are printed to probe for approximately 500 different antibodies to produce a functional proteomic profile of each sample.
Advantages of RPPA and Functional Proteomics
Studies of complex diseases such as cancer have shown that genetic alterations do not account for all causes of the disease. Changes in protein levels and structure have also been shown to play critical roles in tumor development and progression. In cancers, several genetic and epigenetic changes are often required for development of the disease. Studying large-scale epigenetic changes such as protein phosphorylation or cleavage will greatly aid in understanding the causes and determining effective treatment of cancers and other complex diseases.
Advantages of the RPPA platform include the following:
- Cost effective: A high throughput approach concurrently tests and quantifies over 1000 samples on a signal slide.
- Sensitive: Applicable to very small sample sizes (ng of protein lysates, detecting attomoles of a specific protein), less than 10 cell equivalents. It detects and quantifies hundreds of different proteins at their expression levels and modification status in 80 µl of cell lysates.
- Quantitative: Serially diluted samples enable detection of proteins in their linear range to reliably determine relative protein levels of all samples.
- Simple: Does not require direct labeling of the sample; samples can be submitted without additional modification
- Versatile: An antibody-based platform can probe for various protein-associated effects including modification of basal protein expression levels, growth factor‐ or ligand‐induced effects, and time resolved responses appropriate for systems biology analyses. It provides information to integrate the consequence of genetic aberrations in cancer, validate therapeutic targets, demonstrate on‐ and off‐target activity of drugs, and evaluate drug pharmacodynamics.
Our Unique Platform
MD Anderson's RPPA Core maintains and improves upon its high quality output by continuously validating and performing quality control checks on all aspects of its RPPA Pipeline, including the following:
- Serial dilutions of each sample to capture the linear antibody-antigen reaction for accurate data analysis
- A continuously developing and highly stringent antibody validation process to select and maintain only the most qualified antibodies for RPPA analysis
- 48 unique cell lysates printed on every slide for quality controls of data generation and analysis as well as for replicates-based normalization to merge RPPA data across different slides
- A combination of various additional quality control processes developed, implemented, and optimized for our RPPA data analysis to provide reliable data to customers: algorithms of spatial correction, quality control of antibody probing, protein loading correction, replicates-based normalization, and quality determination of antibody batches
- An automated program for RPPA Pipeline processes implemented to reduce manual labor, human error, and customer turnaround time
The RPPA Process
The RPPA Core analyzes expression and modification of proteins in cultured cells (human and animal) and tissues, including blood cells (excluding erythrocytes) and patient derived xenografts.
- View our RPPA Description
- Characterize cell signaling networks under different culture conditions
- Determine drug selectivity
- Identify therapeutic targets
- Define regulatory mechanisms in signaling networks, including forward and feedback loops and crosstalks
- Analyze up to 1000 conditions concurrently such as for detailed concentration and time course studies for systems biology approaches
- Classify patient tumors
- Correlate DNA, RNA and protein
- Determine prognosis
- Predict responses to targeted therapies
- Provide pharmacodynamics and biologically relevant dose
- Determine appropriate handling procedures for clinical samples (based on antigen stability analysis)
We currently do not accept FFPE samples for RPPA processing due to the lack of quality and analyzable protein present within this sample type.