shRNA

General shRNA FAQs


MISSION shRNA

What is shRNA?
Do you guarantee knockdown using your clones?
What gene family sets are available?
Why is there always a clone for the 3' UTR region?
Can I search and design my own shRNAs on the TRC "Search for shRNA hairpin sequences page?"
Your MISSION TRC shRNA clones are sequenced after cloning, but are they sequenced routinely after each round of amplification due to the high frequency of recombination that occurs in retroviral vectors?
I am trying to decide between the three types of RNAi available through Sigma-Aldrich®—the bacterial stock, the plasmid DNA, and the lentiviral particle for a research project. For the plasmid DNA, is there a way to multiply the stock we've been given, using PCR or any other method? Also, are there any research papers or links to some literature that allow me to view the data on the genes that have been silenced?
In what formats can the shRNA clones be ordered?
Are there special protocols for transfection of the shRNA plasmids?
What is a TRC Number?
Do I need a license to use this product?
Do I need to purchase a separate license for commercial applications?
How many times can you freeze/thaw glycerol, DNA or viral stocks?
Do you have matching cell bio/PCR products for your shRNAs?
What vehicles have been used to deliver shRNAs?
When will shRNA validation be completed?
How have shRNAs been validated?



What is shRNA?
shRNA is a short-hairpin sequence within the LTR regions of the lentiviral construct. Upon integration of the LTR to LTR area into the host genome, the shRNA is produced by the cell and shuttled out into the cytoplasm where it is cleaved by the enzyme complex Dicer. Once this cleavage is complete, one strand of this double-stranded molecule is loaded into the RISC (RNA Induced Silencing Complex), where is seeks complementary mRNA targets for degradation.

Do you guarantee knockdown using your clones?
Sigma-Aldrich guarantees that upon purchase of the defined clone set quantity on the shRNA detail page for your gene of interest (this varies, but is typically 5 clones), that at least one of those clones for a gene should yield greater than 70% knockdown.

What gene family sets are available?
Currently available in bacterial glycerol stock format
Currently available in lentiviral transduction particle format
We are continually updating our gene panel offerings. They are provided in either glycerol or lentivirus formats. Additional sets will be made available over the coming months. If your gene set of interest is not yet available, please inquire for advanced custom preparation at MISSIONRNAi@sial.com. We can assemble custom gene sets at any time.


Why is there always a clone for the 3' UTR region?
The clones that are against the 3' UTR of the target are included so one could rescue the knockdown phenotype with the introduction of a cDNA clone corresponding to the target. Transcripts from an exogenous cDNA clone would not be susceptible to silencing compared to the endogenous copy. Many publications/review committees require such an experiment to verify the observed phenotype. However, while most target sets have this added feature, not all target sets have a 3' UTR clone.

Can I search and design my own shRNAs on the TRC "Search for shRNA hairpin sequences page?"
The algorithm used to design the MISSION TRC clone library is generally the same as what is found on the TRC page. However, there have been numerous improvements to the design as the library is being cloned by the Broad-TRC. The clones that you find on the public TRC page are not the most current design version. The updated target sets are only available to TRC members and through purchase with Sigma-Aldrich®.

Your MISSION TRC shRNA clones are sequenced after cloning, but are they sequenced routinely after each round of amplification due to the high frequency of recombination that occurs in retroviral vectors?
We do sequence verify clones in a random fashion after copying. We sequence approximately 5% of clones of the last copy made. Our R&D group has not seen any evidence of recombination. The TRC library has been cloned into a backbone that was optimized to avoid these issues.

I am trying to decide between the three types of RNAi available through Sigma-Aldrich—the bacterial stock, the plasmid DNA, and the lentiviral particle for a research project. For the plasmid DNA, is there a way to multiply the stock we've been given, using PCR or any other method? Also, are there any research papers or links to some literature that allow me to view the data on the genes that have been silenced?
The MISSION TRC shRNA library is composed of three formats to give you flexibility and convenience. The purified plasmid DNA format may be used directly for transient transfection assays or used with our lentiviral packaging mix (Product No. SHP001) to produce lentiviral particles. You may further propogate the DNA by transforming the plasmids into competent cells (we recommend GC5 cells) to create bacterial glycerol stocks.

The lentiviral particles offer the most convenience. They may be added directly to cells for stable transduction. You are correct; the cells that are transduced will continue to express the integrated shRNA. The cells may be expanded for many downstream assays.

Below are helpful links where you can find additional validation data. You may also download a copy of the Cell publication highlighting the use of the library in a high-content screen.
Application Data
RNAi Bibliographies

In what formats can the shRNA clones be ordered?
We offer mulitple shRNA clones, usually around five, for both the human and mouse genomes. Oftentimes, other species can be accommodated using this library, and our bioinformatics team can help you through this. Three formats are available for each target clone: bacterial glycerol stock, purified plasmid DNA, and lentiviral particles. For more information on ordering MISSION products, please click here.

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.

Additional protocols may be found on our MISSION Protocols page

What is a TRC Number?
A unique number developed by The RNAi Consortium to identify the TRC Library. A TRC number begins with "TRCN" followed by 10 digits. (e.g. MISSION shRNA TRCN0000030720).

Do I need a license to use this product?
If you purchase MISSION TRC products from Sigma-Aldrich for research purposes, a label license to the Oxford BioMedica patents and Benitec patents is conveyed to you. However, if you obtain lentiviral shRNA vectors from other sources, you will need to purchase licenses to these patents. Licenses are available from Sigma-Aldrich for research use. Please consult your own legal department for opinions regarding freedom to operate.


Do I need to purchase a separate license for commercial applications?
Yes. For commercial uses, you will need to contact OxfordBioMedica and Benitec for separate licenses.


How many times can you freeze/thaw glycerol, DNA or viral stocks?
The DNA and glycerol stocks can accommodate multiple freeze/thaws; although we highly suggest preventing excessive freeze thaws. That said, multiple freeze/thaws for lentivirus stocks can severely affect the functionality of the virus. We suggest aliquoting after the first thaw to maximize the viability in future experiments.


Do you have matching cell bio/PCR products for your shRNAs?
Sigma offers validated, high-quality antibodies for protein detection. In addition, we offer an array of PCR-related products.


What vehicles have been used to deliver shRNAs?
Non-viral delivery: naked, polymer and lipid-based delivery reagents Viral delivery: lentivirus, retrovirus and adenovirus.


When will shRNA validation be completed?
The Broad and Sigma-Aldrich are validating thousands of shRNAs every month, and providing this data for your evaluation on our website. We hope to have this completed sometime in 2010.


How have shRNAs been validated?
SYBR Green qPCR was used to determine relative silencing effect of particular shRNAs.

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



MISSION is a trademark of Sigma-Aldrich. Label License.

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