Genome-Wide Libraries for RNA-Interference

Principle function of human RNAi
Long double-stranded (ds)RNA or hairpin RNA substrates are cut by the protein Dicer into smaller (~21-nucleotide (nt)) small interfering (si)RNAs with 2-nt overhangs at the 3′ ends and phosphate groups at the 5′ ends. Alternatively, siRNA duplexes (19–23 nt) can be introduced into cells, where they are phosphorylated at the 5′ ends by cellular kinases. These small dsRNAs assemble into the RNA-induced silencing complex (RISC), which contains AGO2, Dicer and other cellular factors.  siRNA then forms activated RISC (siRISC) that contains an antisense (guide) strand. Activated RISC finds its target mRNA and uses the antisense strand to guide the cleavage of the target mRNA. RISC is recycled and could carry out several cleavage events.

Human genome RNAi-library:
about 20.000 genes are targeted by corresponding RNAi molecules in triplicates

C. elegans genome RNAi-library:
The whole genome library consists of 16,757 bacterial strains for feeding, which cover 87% of C. elegans genes.

Process Automation:
FreedomEvo-System (Tecan AG) with 384-needle TeMo, 8-needle LiHa (pipetting positive and negative reference RNAi) and incubators for complete automation of transfection of cells in 384well microtiterplates.