siRNA newb - How to go about starting an siRNA project (Nov/17/2010 )
Hi! I am wanting to start a new project using siRNA to knock down a specific protein's expression and then analyze the cell's response. I am totally new at siRNA and nobody currently in the lab has used it either. I am wondering how is the best and cheapest way to go about starting this considering that there is an article already out that used siRNA on the same target. They used the Dharmacon Smartpool siRNA, which is a mixture of four oligonucleotide sequences, and the sequences are provided in the article, but I noticed that the sequences that the article provides aren't complementary. I feel like either something is wrong with the article or I'm not understanding something important. So should I just design my own and validate them? When I go to the website, and look at the predefined siRNA library, while they do give the option of the smartpool design, I don't see the actual sequences that they would be sending me. Sooo...I don't know if I should go ahead and order the smart pool design or use the custom design function. Or design my own and then test them independently instead of pooling them? I'm feeling really overwhelmed with options, and so any guidance from someone who has actually done this would be fantastic!
don't feel overwhelmed at all the options for RNAi. What option to take depends on your budget. First I'd like to point to you the fact that RNAi is quite robust and even randomly picked targets have an over 90% success rate according to Tuschl.
If you don't have a deep pocket, design your own siRNA using some of the online tools such as Invitrogen siRNA design tool called BLOCK-iT RNAi Designer (Choose the siRNA design option). You can just design one or two siRNAs and are almost guaranteed to obtain a functional siRNA. Dahmarcon's pre-designed or custom designed siRNAs tend to be very expensive and are not necessarily superior than self-designed ones.
Besides the siRNAs for your target gene, you need a control siRNA that is either scrambled from your target siRNA or contains unrelated sequence that does not have any significant homology with genome or mRNA sequence of your organism.
There are good arguments to be made for both ideas. While designing your own might be cheaper, and you could potentially obtain an effective knockdown by only using one or two siRNA, there's no guarantee and you might wind up starting over. There is a part of me that says, if someone has already published with a specific product, shown it works, and you can afford it, why wouldn't you use it? Sure, it might be more expensive but you have the guarantee that it will work and you'll be able to ultimately reference that other paper when you publish your work. This is what I am currently doing and the exact reason why I chose the siRNA that I'm working with. Another thing to keep in mind is how stable is your protein of interest? I was trying to siRNA an extremely stable protein and could only obtain acceptable knockdown with the Smartpool siRNA after two transfections (24 hours apart). The more stable your protein, the more difficult it is to make it go away. Also, what do you mean by the sequences aren't complementary? They shouldn't be complementary to each other and, unless you are aligning to the genomic sequence (not mRNA) you may not find the targeted sequence since many siRNAs are designed to the 5'UTR or introns. You do always have the option of ordering the published sequences one at a time and testing them for effectiveness. I ordered one of four published siRNAs from Invitrogen with the Stealth modification and it works great!! This may allow you to save some money and yet still use the published paper as a reference.
Hi! Thank you for your suggestions and input. My boss would really prefer to just replicate what these other people have done, that way if(/when) it doesn't work, at least we can be relatively sure that it's me and not the siRNA I realized my mistake with the complementary business--total newb mistake. The two strands that were published ARE complementary, except for the 2bp overhang--somehow I missed this detail for a long time. Once I figured this out I simply called the company and asked them if the siRNA that was published were availiable from their predesigned selection. I told them the sequence, and they told me the catalog number--so all is well!