The siRNA libraries:
The Drug Discovery, Development, and Delivery Core has available eight libraries from Invitrogen. Each library has three separate siRNAs for each gene in the collection. Primary screening is done with each of the three siRNAs combined into one reaction. The single siRNAs can be used for confirmation in a secondary screen. The libraries are arrayed on 96 well plates. Each plate has the following controls, low, medium, high GC scrambled siRNAs, p53 siRNA, and fluorescent siRNA and seven wells for user defined controls.
The core provides the nucleofection kit, tips, 96 well plates and other consumables. The investigator is responsible for media, and reagents for screening. The core will perform the transfection/nucleofection and the screen.
# plates (96 well)
siRNA Kinase (636)
siRNA Phosphastase (237)
siRNA Nuclear Receptor (48)
siRNA Nuclear Receptor Cofactor (280)
siRNA Ion Channel (326)
siRNA Non-olfactory GPCR (412)
siRNA Protease (366)
siRNA Ubiquitin Protease (53)
Lo GC Neg
Med GC Neg
Hi GC Neg
AllStars Hs CELL DEATH CONTROL siRNA
The Core now has available a new positive control for transfection. AllStars Hs Cell Death Control siRNA is a mixture of siRNAs targeting genes that are essential for cell survival. Knockdown of these genes induces a high degree of cell death in most cell lines. Transfection efficiency can be quickly estimated 24–96 hours after transfection. It can be utilized for transfection optimization and as a positive control when screening (learn more about AllStars)
The core uses Amaxa’s nucleofection technology (now part of Lonza) for screening of siRNA libraries. Nucleofection is an electroporation method that introduces siRNA/plasmids into the nucleus of cells. In order to use nucleofection an appropriate nucleofection program must be selected. This can be done by looking up your cell line on Lonza’s website (www.lonza.com). Make sure the program number is for the 96 well shuttle system. If your cell line is not present in Amaxa’s database, Amaxa sells a nucleofector optimization kit that can be used to choose the correct program. Depending on cell density and transfection efficiency the siRNAs can be run replicates of three or four. The fees for nucleofection are listed below.
In addition to nucleofection, the core utilizes lipofection for siRNA transfection. For lipofection of cells the core suggests Lipofectamine RNAiMAX from Invitrogen using their reverse transfection protocol. The manual for RNAiMAX has appropriate starting concentrations for different sized plates. A Sample protocol is available upon request for a typical assay set up in the core. Since the concentration of siRNA used in the screen is minimal, it must be diluted first. This means that each siRNA can be run up to 9 times from one dilution. The core suggests considering running the siRNAs in triplicate or in multiple cell types to get the most value out of the dilution. The core can provide Lipofectamine RNAiMAX at a cost of $0.40/ul or the investigator can supply it to the core.
Fee includes optimization of lipid transfection using RNAiMax lipofectomine and All Stars Cell Death control and scrambled siRNA.
The core has available a molecular devices plate reader that is capable of doing fluorescence, absorbance, luminescence, time resolved fluorescence, and fluorescent polarization. Endpoint, kinetic, and spectrum reads are possible. The flex unit allows addition of reagents to the wells immediately before reading the wells allowing us to do assays such as flash luminescence. Use of the plate reader is included in the base price of siRNA screens.
The core has available an EPmotion 7050 liquid handling robot. The core uses this robot for dispensing the chemical library for assays. The robot can also be used for adding other reagents required for the assay. The core can assist in setting up the best way to do this. There is an additional charge for consumables used with the robot. The EPMotion has a wide range of capabilities including but not limited to performing dilution series, repeat pipetting, delivery of as little as 2 ul of sample, transfer of reagents from tubs, microcentrifuge tubes, 96 and 384 well plates to the destination plates.
The 96 well Shuttle System is a 96 well nucleofector module from Amaxa (now part of Lonza). It will transfect cells in 96 well plates cells are then transferred to tissue culture plates to recover and analyze. Nucleofection protocols can be individualized for each well or the same protocol used for the whole plate. This system is designed for difficult to transfect cell types including primary cells.