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
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
Early life stress (ELS) is highly associated with development of psychopathology
and mood disorders in adulthood. Genetic studies have identified variation in the gene calcium voltage-gated channel subunit alpha1C (CACNA1C) to increase risk for several psychiatric disorders. This poster assessed the expression of Cacna1c following prepubertal stress.
18th International Conference on Pharmaceutics & Novel Drug Delivery Systems
May 27 - May 28, 2019