Strengths
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. 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 . RPMAs have increased sensitivity and are capable of detecting proteins in the picogram range . Some researchers have even reported detection of proteins in the attogram range. This is a significant improvement over protein detection by ELISA, which requires microgram amounts of protein (6). The increase in sensitivity of RPMAs is due to the miniature format of the array, which leads to an increase in the signal density (signal intensity/area) coupled with tyramide deposition-enabled enhancement. The high sensitivity of RPMAs allows for the detection of low abundance proteins or biomarkers such as phosphorylated signaling proteins from very small amounts of starting material such as biopsy samples, which are often contaminated with normal tissue . Using laser capture microdissection lysates can be analyzed from as few as 10 cells, with each spot containing less than a hundredth of a cell equivalent of protein.
A great improvement of RPMAs over traditional forward phase protein arrays is a reduction in the number of antibodies needed to detect a protein. Forward phase protein arrays typically use a sandwich method to capture and detect the desired protein. This implies that there must be two epitopes on the protein (one to capture the protein and one to detect the protein) for which specific antibodies are available. Other forward phase protein microarrays directly label the samples, however there is often variability in the labeling efficiency for different protein, and often the labeling destroys the epitope to which the antibody binds. This problem is overcome by RPMAs as sample need not be labeled directly.
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. This allows for the quantification of differences in protein levels across all samples. 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. 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. A problem that is encountered with tissue microarrays is antigen retrieval and the inherent subjectivity of immunohistochemistry. Antibodies, especially phospho-specific reagents, often detect linear peptide sequences that may be masked due to the three-dimensional conformation of the protein. This problem is overcome with RPMAs as the samples can be denatured, revealing any concealed epitopes.
Read more about this topic: Reverse Phase Protein Lysate Microarray
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