[1] 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.
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.
[4] 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.
PMAs immobilize individual purified and sometimes denatured recombinant proteins on the microarray that are screened by antibodies and other small compounds.
[1] Aliquots of the lysates are pooled and resolved by two-dimensional single lane SDS-PAGE followed by western blotting on a nitrocellulose membrane.
[1] The lysates are then printed onto either nitrocellulose or PVDF membrane coated glass slides by a microarrayer such as Aushon BioSystem 2470 or Flexys robot (Genomic solution).
[10] The membrane coated glass slides are commercially available from several different companies such as Schleicher and Schuell Bioscience (now owned by GE Whatman www.whatman.com),[9] Grace BioLabs (www.gracebio.com), Thermo Scientific, and SCHOTT Nexterion (www.schott.com/nexterion).
[14] In this study, they report the use of a dual dye-based approach that can effectively double the number of endpoints observed per array, allowing, for example, both phospho-specific and total protein levels to be measured and analyzed at once.
[1] These programs quantify signal intensities at each spot and use a dose interpolation algorithm (DI25) to compute a single normalized protein expression level value for each sample.
The greatest strength of RPMAs is that they allow for high throughput, multiplexed, ultra-sensitive detection of proteins from extremely small numbers of input material, a feat which cannot be done by conventional western blotting or ELISA.
[1][9] The small spot size on the microarray, ranging in diameter from 85 to 200 micrometres, enables the analysis of thousands of samples with the same antibody in one experiment.
Another strength of RPMAs over forward phase protein microarrays and western blotting is the uniformity of results, as all samples on the chip are probed with the same primary and secondary antibody and the same concentration of amplification reagents for the same length of time.
Furthermore, printing each sample, on the chip in serial dilution (colorimetric) provides an internal control to ensure analysis is performed only in the linear dynamic range of the assay.
[4] Optimally, printing of calibrators and high and low controls directly on the same chip will then provide for unmatched ability to quantitatively measure each protein over time and between experiments.
Antibodies, especially phospho-specific reagents, often detect linear peptide sequences that may be masked due to the three-dimensional conformation of the protein.
[1] To overcome this issue, two open resource databases have been created to display western blot results for antibodies that have good binding specificity within the expected range.
In addition, the technique has now been brought into clinical trials for the first time whereby patients with metastatic colorectal and breast cancers are chosen for therapy based on the results of the RPMA.