siRNA Screening: Development of Hit Stratification Strategies
Poster Mar 10, 2015
Žaklina Strezoska, Annaleen Vermeulen, Emily M. Anderson, Anja Smith, Devin Leake
While synthetic siRNA libraries are powerful tools for functional genomic screens, off-target effects mediated by siRNA seed interactions with the 3' UTR of unintended targets can result in false positives. Given the frequency of off-target effects in some assays, the development of hit validation/stratification strategies is imperative. In the following study we have compared two strategies for identification of high confidence hits: 1) a multiple reagent approach where two or more individual siRNAs induce the same phenotype and 2) a chemical modification approach where hit confirmation is achieved using pools of siRNA that contain specificity enhancing modifications. A comparison of these two strategies (using a collection of primary hits generated from a cell viability screen) reveals significant overlap between the high confidence hits identified. However, for low confidence hits, i.e. where a single siRNA induces a phenotype, the concern is that an important hit will be missed. To determine if the phenotype is due to gene targeting or a seed-mediated off-target effect, a chimeric approach was used whereby a gene-specific seed sequence is introduced into a non-targeting siRNA scaffold. Together, these data provide well-defined approaches for prioritization of hits derived from RNAi screens.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE