Genomic Screening Techniques

Genome-Wide Libraries for RNA-Interference
The liposome reagent mediated transfection of cells with RNA-oligomers is the most expensive type of screening supported by our platform. The libraries are provided in 384well format with last two columns empty for control transfections with reference RNA. About 200 microtiter plates in 384well format are used for independent transfections in triplicate to identify stable readout. When dual luciferase reporter cell lines are used the costs for screening may start already with 36.000 Euro for luciferase substrate plus costs for liposome reagents. Therefore establishing  GFP coupled reporter systems may reduce costs significantly. The high costs essentially require to acquire apropiate funding, sufficient time for assay set up for HTS (up to 6 months including automated object identification in High Content Screens with automated microscopes). Do not hesitate to contact the Screening Unit to estimate costs, which really exceed the normal budget of a research group.

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

Mouse genome RNAi-library:
also 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.