Abstract
A gene expression-based siRNA screen was used to evaluate functional similarity between genetic perturbations to identify functionally similar proteins. A siRNA library (siGenome library, Dharmacon) consisting of multiple siRNAs per gene that have been pooled in to one well per gene was arrayed in a 384-well format and used to individually target 14,335 proteins for depletion in HCT116 colon cancer cells. For each protein depletion, the gene expression of eight genes was quantified using the multiplexed Affymetrix Quantigene 2.0 assay in technical triplicate. As a proof of concept, six genes (BNIP3, NDRG1, ALDOC, LOXL2, ACSL5, BNIP3L) whose expression pattern reliably reflect the disruption of the molecular scaffold KSR1 were measured upon each protein depletion. The remaining two genes (PPIB and HPRT) are housekeeping genes used for normalization. The gene expression signatures from this screen can be used to estimate the functional similarity between any two proteins and successfully identified functional relationships for specific proteins such as KSR1 and more generalized processes, such as autophagy.