siRNA Screening

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.

  

Library
# plates (96 well)
siRNA Kinase (636)
7.95
siRNA Phosphastase (237)
2.9625
siRNA Nuclear Receptor (48)
0.6
siRNA Nuclear Receptor Cofactor (280)
3.5
siRNA Ion Channel (326)
4.075
siRNA Non-olfactory GPCR (412)
5.15
siRNA Protease (366)
4.575
siRNA Ubiquitin Protease (53)
0.6625

 
Sample plate Map

 
1
2
3
4
5
6
7
8
9
10
11
12
A
VHS50172
HSS114301
VHS41387
HSS121096
HSS104407
HSS112280
HSS120796
VHS41680
HSS112847
HSS124795
VHS50498
HSS111095
B
VHS40462
HSS108515
VHS50108
HSS135373
HSS100625
VHS50889
VHS40565
VHS40547
HSS103336
HSS114410
VHS40805
VHS40726
C
HSS121928
HSS128033
VHS41062
HSS107350
HSS118945
HSS102071
HSS104617
HSS103745
HSS106451
HSS118418
VHS50980
HSS121219
D
HSS119632
HSS118097
VHS50025
HSS123398
HSS103747
HSS102823
HSS106802
HSS115375
VHS50791
HSS140560
HSS115177
HSS126106
E
HSS100981
HSS107941
HSS107791
HSS134968
HSS102388
VHS50146
VHS40558
HSS108805
HSS128134
HSS101329
VHS40662
HSS108569
F
HSS139183
VHS40279
HSS136604
VHS50354
HSS108469
HSS119066
HSS130463
HSS122732
HSS108802
HSS121267
HSS101731
HSS112985
G
VHS50035
HSS108511
HSS122611
HSS132254
HSS105821
HSS138352
HSS113734
HSS120130
HSS101098
HSS119266
HSS117751
HSS113470
H
Fluorescent Ctrl
 User Control
User Control
User Control
User Control
User Control
User Control
User Control
p53
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)

 

Nucleofection: 

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.

 
Lipofection:

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.

 
Synergy Analysis ****NEW****
 
The core now has available BioSoft's Calcusyn Software.  This software allows one to determine whether two drugs act synergystically or not.  For new users the Core provides a training session that covers experimental design, data entry and analysis of results ($11.78).  After training, investigators may arrange to do their own analysis ($3 per combination) or have the Core perform the analysis ($10.77).  We also have the capability to run your synergy assay.  Please contact us to discuss pricing.
 

Transfection Optimization:

Fee includes optimization of lipid transfection using RNAiMax lipofectomine and All Stars Cell Death control and scrambled siRNA. 

 

 
Equipment:
 
Flex Station3 Plate Reader:

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.

 
Liquid Handling Robot:

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.

 
96 well Shuttle System:

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.