Hello,
I am planning to design a ssRNA molecule that I will use as a microRNA to block the translation of GFP. I will use this as a positive control for transfection.
My question is, what factors should I bear in mind when designing the ssRNA molecule complementary to the mRNA of GFP? Should the G/C content be around 55% like when you design primers for PCR?
I guess using an online tool for the design of primers would be good as well to design an artificial microRNA?
Best,
Adrian
If you are simply looking for an online tool, then i will suggest you Web microRNA Designer. Please go through the link.
http://wmd3.weigelworld.org/cgi-bin/webapp.cgi?page=Help#procedure
Invitrogen offers a miRNA-RNAi system for vector-based expression of artificial miRNAs. They have a design tool on their homepage which is free and can be used without creating an account. Obviously, the generated oligonucleotides carry the overhangs for their Vectors, so you might need to change them a little. The System is called BLOCK-iT™ Pol II miR RNAi Expression Vector System and the tool is found at https://rnaidesigner.thermofisher.com/rnaiexpress/. While of course their homepage aims for selling their products, nontheless both design and information on the system should be appliable to your problem.
Curiosity gets the best of me, cause I have to ask:
Why go through all that trouble when you can get RNAi controls for GFP, pre-designed and readily available ? Case in point; Thermofischer cat#AM4626 (siRNA).
Cheers
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