A reverse phase protein lysate microarray (RPMA) is a protein microarray designed as a dot-blot platform that allows measurement of protein expression levels in a large number of biological samples simultaneously in a quantitative manner when high-quality antibodies are available. Technically, minuscule amounts of (a) cellular lysates, from intact cells or laser capture microdissected cells, (b) body fluids such as serum, CSF, urine, vitreous, saliva, etc., are immobilized on individual spots on a microarray that is then incubated with a single specific antibody to detect expression of the target protein across many samples. A summary video of RPPA is available. One microarray, depending on the design, can accommodate hundreds to thousands of samples that are printed in a series of replicates. Detection is performed using either a primary or a secondary labeled antibody by chemiluminescent, fluorescent or colorimetric assays. The array is then imaged and the obtained data is quantified. Multiplexing is achieved by probing multiple arrays spotted with the same lysate with different antibodies simultaneously and can be implemented as a quantitative calibrated assay. In addition, since RPMA can utilize whole-cell or undissected or microdissected cell lysates, it can provide direct quantifiable information concerning post translationally modified proteins that are not accessible with other high-throughput techniques. Thus, RPMA provides high-dimensional proteomic data in a high throughput, sensitive and quantitative manner. However, since the signal generated by RPMA could be generated from unspecific primary or secondary antibody binding, as is seen in other techniques such as ELISA, or immunohistochemistry, the signal from a single spot could be due to cross-reactivity. Thus, the antibodies used in RPMA must be carefully validated for specificity and performance against cell lysates by western blot. RPMA has various uses such as quantitative analysis of protein expression in cancer cells, body fluids or tissues for biomarker profiling, cell signaling analysis and clinical prognosis, diagnosis or therapeutic prediction. This is possible as a RPMA with lysates from different cell lines and or laser capture microdissected tissue biopsies of different disease stages from various organs of one or many patients can be constructed for determination of relative or absolute abundance or differential expression of a protein marker level in a single experiment. It is also used for monitoring protein dynamics in response to various stimuli or doses of drugs at multiple time points. Some other applications that RPMA is used for include exploring and mapping protein signaling pathways, evaluating molecular drug targets and understanding a candidate drug's mechanism of action. It has been also suggested as a potential early screen test in cancer patients to facilitate or guide therapeutic decision making. Other protein microarrays include forward protein microarrays (PMAs) and antibody microarrays (AMAs). PMAs immobilize individual purified and sometimes denatured recombinant proteins on the microarray that are screened by antibodies and other small compounds. AMAs immobilize antibodies that capture analytes from the sample applied on the microarray. The target protein is detected either by direct labeling or a secondary labeled antibody against a different epitope on the analyte target protein (sandwich approach). Both PMAs and AMAs can be classified as forward phase arrays as they involve immobilization of a bait to capture an analyte. In forward phase arrays, each array is incubated with one test sample such as a cellular lysate or a patient's serum, but multiple analytes in the sample are tested simultaneously. Figure 1 shows a forward (using antibody as a bait in here) and reverse phase protein microarray at the molecular level.
Experimental design and procedure Depending on the research question or the type and aim of the study, RPMA can be designed by selecting the content of the array, the number of samples, sample placement within micro-plates, array layout, type of microarrayer, correct detection antibody, signal detection method, inclusion of control and quality control of the samples. The actual experiment is then set up in the laboratory and the results obtained are quantified and analyzed. The experimental stages are listed below:
Sample collection Cells are grown in T-25 flasks at 37 degree and 5% CO2 in appropriate medium. Depending on the design of the study, after cells are confluent they could be treated with drugs, growth factors or they could be irradiated before lysis step. For time course studies, a stimulant is added to a set of flasks concurrently and the flasks are then processed at different time points. For drug dose studies, a set of flasks are treated with different doses of the drug and all the flasks are collected at the same time. If a RPMA containing cell fraction lysates of a tissue/s is to be made, laser capture microdissection (LCM) or fine needle aspiration methods is used to isolate specific cells from a region of tissue microscopically.
Cell lysis Pellets from cells collected through any of the above means are lysed with a cell lysis buffer to obtain high protein concentration.
Antibody screening Aliquots of the lysates are pooled and resolved by two-dimensional single lane SDS-PAGE followed by western blotting on a nitrocellulose membrane. The membrane is cut into four-millimeter strips, and each strip is probed with a different antibody. Strips with single band indicate specific antibodies that are suitable for RPMA use. Antibody performance should be also validated with a smaller sample size under identical condition before actual sample collection for RPMA.
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