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shRNA

How to Set Up Experiments

 
MISSION shRNA

What controls do you recommend?
When conducting experiments using MISSION TRC shRNA constructs, the proper controls should be a key element of your experimental design to allow for accurate interpretation of knockdown results. All controls are available in both purified plasmid DNA and lentiviral particle format.

 

In addition, MISSION TurboGFP Control Transduction Particles (SHC003V) can serve to optimize transduction efficiency when using a cell line for the first time.

Sigma's recommended controls for any shRNA experiment are provided in the Control Selection table and are closely aligned with the controls suggested in the Nature Cell Biology editorial.1


Why doesn’t the non-target control vector affect gene expression?
The non-target control vector (SHC002, SHC002V).has at least 5 bp mismatches within the shRNA to any known human or mouse genes, limiting its ability to target any human or mouse genes. Please refer to the product listing for hairpin sequence and vector map.


How many cells can I infect with the amount of lentivirus provided?
The amount of cells that can be infected depends upon the cell line being used. For HeLa, A549 and HEK293T cells we have used 4 µl (1 x 106 TU/ml via p24 assay) for each well containing ~16,000 cells in a 96-well plate. Primary or other difficult cells may require more lentiviral supernatant, but even at a 10 µl level, it would give you enough for 20 x 96-well reactions. We suggest decreasing the number of cells plated to increase the Multiplicity of Infection (MOI) if necessary. Also, performing a limiting dilution titer on your cell line will give you the optimal amount of viral particles needed for each assay.


Which cell types can be transduced with the lentiviral particles?
We have listed cell types that have been successfully transduced with the pLKO.1-puro lentiviral constructs.


Are there special protocols for transfection of the shRNA plasmids?
No. You may use your current method of transfecting plasmid DNA. If you do not currently transfect plasmid DNA, we recommend ESCORT™ V Transfection Reagent.


Contact Us
For questions about the library, pricing and quotes or other concerns, please e-mail us at: MISSIONRNAi@sial.com.

MISSION is a registered trademark of Sigma-Aldrich Co. LLC Label License.

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