Hi everyone.
I'm working with the proteins hnRNP A1 and HuR (or ELAV-L1) and I need to silence the expression of the endogenous proteins to analyze the effect of over-expressed mutagenic proteins. In order to this, I need to design siRNAs that binds complementary to non coding regions (5'NCR or 3'NCR) of the messenger. There is only one comercial siRNA available for each proteins, which will be probably not enough to silence them. I'm thinking about ordering designed siRNAs from Dharmacon, but I don't know which considerations should I have... i.e. how I determine the complete 5' or 3' NCRs of a gene, to design the siRNAs? Should I consider the regions immediately before the ATG and after the stop codon? and how many siRNAs should I design to try as a mix with the comercial one?
Thank you very much!
Aldo.
Hi,
I have used PSICOLIGOMAKER v1.5 for my work. It is based on a criteria defined by Vantura et al, 2014 to design an effective siRNA sequence against the sequence of interest. Hope it helps.
Hi Aldo
Besides Sheerien's post, you probably might give a try to LNA Gapmers. Mechanism of knocking-down is slightly different because it may involve RNAse H, but they are pretty good in silencing ncRNAs. I never tried them in ncRNA regions of coding transcripts, but the LNA will ensure a more specific and tighter binding to the target than the classical RNAi oligo.
Gapmers were sold by Exiqon. Unfortunately Exiqon has been purchased by Qiagen, and now the orders are pretty much more difficult than before.
Best
Hi Aldo,
5'NCR or 3'NCR for the gene of your interest you may see at Enseble (transcript view). It is believed (if you design siRNA yourself) to try at least 3-4 targets within the coding area. Additionally you may try to play with
- oligo- composition: either DNA, RNA, or DNA/RNA (U for T)
- overhanging ends (sites for RISC)
- 3' end protection
Methodes for target choice do not differ much from those methodes applied for common primer sites pick up.
Good luck!
Thank you very much Sheerien, Francisco and Andrey! it helped a lot.
Best,
Aldo.
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