High-Throughput Sirna Screening

High-Throughput siRNA Screening

The CCIB DNA Core has acquired the Silencer Select Human Genome siRNA Library V4 (Part Numbers 4397922, 4397924, and 4397923), which targets a genome-scale collection of 21,585 human genes. Our Laboratory Automation group now provides a high-throughput screening service utilizing a pooled version of the library. The pooled library consists of a total of 248 plates (96-well format), with three unique, non-overlapping siRNAs per well targeting the same gene.

The high-throughput screening process at the CCIB DNA Core is designed to be a highly collaborative endeavor between our Laboratory Automation group and the principal investigator/researcher. In our investigator-assisted model, the researcher is responsible for i.) the development, optimization and validation of the screening assay in a 96-well format with positive and negative controls and ii.) for providing cultured mammalian cells - plated in 96-well format - throughout the screening process (all performed at researcher’s home institution). We perform a pilot screen to confirm feasibility of the screen and then continue onto the rest of the genome screen, including data analysis and top hit identification (at our core facility). Should the pilot screen yield unsatisfactory results, the researcher will engage in further assay optimization at his/her home institution.

Screening System Description:

·  Assay plates contain 7.5 pmol of siRNA (2.5 pmol per individual siRNA) in a total volume of 3 ul (96-well format).

·  siRNA transfections of cultured mammalian/human cells are performed in duplicate.

·  Our well-established in-house HTS assay system is based on the Gaussia princeps luciferase enzyme (GLuc) which has considerable advantages over other luminescent reporters:

Ø  The enzyme uses an inexpensive substrate (coelenterazine) and does not require ATP as a cofactor.

Ø  Upon expression, the reporter molecule is secreted into the cell medium and allows direct signal measurement (one-step protocol, without cell lysis). The luminescence is proportional to the amount of enzyme produced, which in turn, reflects the level of transcription and post-transcription regulation.

Ø  A single well (i.e. single transfection) can be used for multiple time points over an extended time period.

Ø  GLuc generates over 1000-fold higher bioluminescent signal intensity compared to firefly luciferase from Photinus pyralis (FLuc) and Renilla reniformis (RLuc).

Ø  We are using a GLuc variant that exhibits prolonged glow kinetics (no burst kinetics as the native activity).

Ø  Our optimized in-house assay buffer confers enhanced signal stability, which allows the use of the assay in high-throughput format.

Please note: Free consultation for the construction of the required GLuc-based reporters will be provided.

·  To measure gene silencing and/or knockdown effectiveness, luminescence read-outs will be performed using the TopCount NXT plate reader.

·  The identities of the top hit clones are determined by statistical analysis.

Customized Screening Projects:

Our experienced team can work with you to adapt your desired assay system to high-throughput screening (HTS).

If you are interested in our siRNA screening service, please contact us to schedule an introductory consultation.