siRNA screening at the functional level using a novel nano-immunoassay platform

A53 The invention of small interfering RNA (siRNA) technology has provided a tool for understanding the function and relationships of proteins as well as strategies for drug development in disease. These tools are culled from libraries of potential candidate molecules through labor intensive screenings, usually employing western blot analysis of target protein levels. This approach becomes difficult if the target protein is expressed in small amounts or in rare cells. Here we present a novel nano-immunoassay platform (Firefly TM ) that has advantages over conventional approaches because it is automated, is extremely sensitive and is capable of analytically measuring protein from a few hundred cells. Neuronal growth factor (NGF) -stimulated neurons were treated with a panel of siRNA’s targeted at disrupting the NGF catalytic receptor trkA. siRNA’s that were specific for the trkA suppressed downstream ERK signaling. Control siRNA’s, which were targeted at proteins not involved in this pathway, exhibited no effect on ERK. The most striking finding of this study was that the effect of this siRNA was on the dual phosphorylation of ERK and not on the single phospho- forms. This type of result shows not only the usefulness of Firefly to measure the effectiveness of siRNA’s on target proteins, but also provides scientific insight into the functions of the proteins targeted (in this case: that trkA is responsible for the second phosphorylation event of ERK in NGF stimulation, but not the first).