Low hit validation rate for Dharmacon siGENOME screens
Good experimental design is important when validating hits from RNAi screens.
Clearly compensating
Genetic compensation by transcriptional adaptation is a process whereby knocking out a gene (e.g by CRISPR or TALEN) results in the deregulation of genes that make up for the loss of gene function.
Pooling only 4 siRNAs increases off-target effects
Low-complexity pools (with 4 siRNAs per gene) should thus lead to overall stronger off-target effects than single siRNAs.
Citations of our Nucleic Acids Research Paper
Our 2014 Nucleic Acids Research paper provides an excellent overview of the siPOOL technology. Google Scholar shows that our paper has been cited 64 times.
Performing target validation well
This blogpost describes issues encountered in target validation and how to safeguard against poor reproducibility in RNAi experiments.
Low complexity pooling does not prevent siRNA off-targets
Low-complexity siRNA pooling (e.g. Dharmacon siGENOME SMARTpools) does not prevent siRNA off-targets. It may in fact exacerbate off-target effects. Only high-complexity pooling (siPOOLs) can reliably ensure on-target phenotypes.
What is the probability of an siRNA off-target phenotype?
Conventional siRNAs have a high probability of giving off-target phenotypes. siRNA off-target effects can be reduced by using more specific reagents or narrowing the assay focus (to reduce the number of relevant genes).
5 factors to consider in multi-gene targeting RNAi screens
Effective functional genomic screening depends on a variety of factors that need to be simultaneously addressed to obtain meaningful results. A recent Cell Reports paper demonstrates this by taking a holistic approach to siRNA screening with the use of multi-isoform/multi-gene targeting to address redundant paralogs and pathways in cancer cells.
Is it important to avoid microRNA binding sites during siRNA design?
To address the question of whether one should avoid microRNA binding sites during siRNA design, we examined whether removing siRNAs that share seeds with native microRNAs would reduce the dominance of seed-based off-target effects in RNAi screening.
Disrupting lncRNA function with siPOOLs (RNAi), antisense oligos and CRISPR
This blogpost covers methods used in the disruption of lncRNA function. Specifically focusing on RNA interference (with siPOOLs), antisense oligos, and CRISPR approaches. Challenges faced with these approaches are addressed.
