I have little experience in siRNA designing....
1) I looked at literature where some time only one sequence is mentioned and sometime sense and antisense sequence...Further sometimes ended with TT and while other dTdT...so m quite confuse..so how can I find correct sequence for siRNA ordering based on literature
2)Further if there is some online tool to design the siRNA against any novel gene not mentioned in the literature.
Any kind of help and suggestions are really appreciated.
Thanks a lot in advance
BLOCK-iTTM RNAi designer
https://rnaidesigner.invitrogen.com/rnaiexpress
you may try this one...
Dear Yong & Aref thanks for the links...but which region would you prefer to knock in ORF or 3'/5' UTR?
further any recommendations for GC Content?
For ordering I agree about getting the top candidate siRNA duplexes from a reputable vendor. However, the most important step is to prove if any of those work or more money should be spent. Oftentimes qPCR or Western blot are tricky screening strategies so new ones need to be implemented. For MINT, the mouse Msx2 interacting nuclear target now called Spen, which as a 10.7+ Kbp of coding sequence, I tried >15 duplexes until finding a good silencing one. This is described in my JBC paper (2005) where our strategy was to create a GFP-chimera of my gene of interest, clone it, target it by cotransfect it with known & excellent anti-GFP duplexes as positive controls. Then by microscopy you screen your candidates duplexes for a signal mimicking the anti-GFP silencing effect. - There are more quantitative approaches like targeting a complementation subunit (say, alpha) necessary for reconstituting a bioluminescence complex (alpha + beta). So if you create a fusion protein of your gene of interest with the alpha subunit, cotransfect it with the beta subunit plasmid plus the anti-alpha siRNA, bioluminiscence should be low (+ control), scrambled siRNA should be high. Screening your siRNAs would follow a similar approach and a good silencing duplex should have low luminiscence counts. Confirmation by qPCR or Western blot comes next.
it's a prefer :
ORF could be better because it could be more specific ... random repeat sequences can exist in UTRs
Or it could be already designed, e.g., Ambion has a big database of already designed and sellable siRNAs (ab. 200.000): http://www.lifetechnologies.com/pl/en/home/life-science/rnai/synthetic-rnai-analysis.html
similarily Sigma or other firms.
this one
https://sispotr.icts.uiowa.edu/sispotr/index.html;jsessionid=E43127B3DC6A26F8F20309B74BBDE2C1
to avoid/minimize off target
RNAi consortium has numerous validated shRNA sequences ready to use. Check it out
http://www.broadinstitute.org/rnai/trc
Best
Suneel
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques