The objective of the RNAi Core is to provide convenient, cost effective services for siRNA/shRNA design, siRNA/shRNA delivery into target cells and knockdown validation to City of Hope investigators.
The primary core services are:
- siRNA/shRNA design
- Optimized siRNA/shRNA delivery into target cells using
-Cationic lipids or
- Screening of siRNA/shRNA libraries
- Quantitative PCR
The RNAi laboratory will design 27mer siRNAs based on the gene accession number provided to the core. A minimum order of two siRNAs is recommended.
One of two methods is employed to transfect siRNAs into target cells:
- Cationic lipids that are optimized for siRNA transfection or
- Electroporation using an Amaxa Nucleofector Device for efficient transfection of primary and difficult-to-transfect cell lines
Cy3-labeled siRNAs are used to initially monitor (by FACS) and optimize the transfection efficiency. Quantitative PCR is used to detect the siRNA-induced gene silencing.
The core laboratory will produce shRNA expressing lentiviral vectors, determine the virus titer and establish stable cell lines after the required IBC/OSBC approval has been obtained by the principal investigator.
RNA and DNA samples are accepted. The core also accepts cell pellets and performs the RNA or DNA isolation. The qPCR assays are run in triplicates using gene-specific probes (Roche Universal Probe Library) for all assays.