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shRNA

Stable Knockdown Cell Lines

Knockdown cell lines are particularly useful in gene function analysis, target discovery, target validation, assay development and compound screening. Generating these knockdown cell lines is most readily accomplished by using lentivirus harboring an shRNA of interest. Transduction with lentivirus has become commonplace, and many researchers prefer to develop their lines in-house. However, for those labs looking to jump-start their RNAi screens, partnering with an established RNAi company may be an expeditious means to hasten your research.

SIRION BIOTECHSigma-Aldrich, in partnership with Sirion Biotech (www.sirion-biotech.de), has developed a select RNAi service team that will generate customer selected shRNA knockdown cell line models. Allow our experienced staff develop your RNAi model of interest. The customized cell lines, produced by Sirion, are based on Sigma’s MISSION® shRNA libraries, a highly functionally validated RNAi library developed by The RNAi Consortium (TRC). Customers will be able to select from either the TRC1 or TRC2 libraries, offered exclusively through Sigma-Aldrich. Initially, this service will only be made available to the German Life Science research community, but may be available worldwide in the near future.

Start your project with us today (contact us). A project begins with an evaluation of the MISSION TRC collection, consisting of 210,000+ shRNAs targeting 38,000+ human and mouse genes, to identify all clones which target your gene of interest. Afterward, Sirion immediately moves into optimizing transduction conditions for your chosen cell line. Sirion then continues with an optimized transduction of the specific shRNA lentivirus clones, generating stable, knockdown cell lines after propagating under puromycin selection. Once a cell line has been generated, knockdown levels are determined via qRT-PCR. The cell lines, along with a comprehensive data sheet explaining the knockdown analysis and propagation instructions are sent directly to the customer to begin using immediately.


Validated shRNA Cell Lines

Cos-1 cells were transduced with lentivirus particles with four different shRNA clones against CYP26A1. Cells were subjected to puromycin antibiotic selection leading to cell death of control cells and bulk cell cultures of transduced cells (A). Cultures of the different transductants were analyzed for best mRNA knock down by qRT-PCR using SYBR® Green I detection (B) showing that two sequences, shRNA #2 and shRNA #4, generated excellent silencing efficiencies of 95% and 94%, respectively.

Figure A

shRNA #1
shRNA #1
shRNA #2
shRNA #2
Non-Target Control
Non-Target Control
     
shRNA #3
shRNA #3
shRNA #4
shRNA 4
Unmodified Cos-1 Cells
Unmodified Cos-1 Cells

 



Figure B



Additional Services
- Monoclonal Cell Line Development and Analysis
- Probe-based qRT-PCR Validation (SYBR® Green I qRT-PCR is our standard offering)
- Western Validation

Contact Us
We look forward to speaking with you about your specific project needs.
Please email us at MISSIONRNAi@sial.com to expedite your RNAi research today!