QUESTION-CAPLE OF siRNAs sequences - (Sep/12/2010 )
I used 2 different siRNAs sequences to knockdown the same mRNA.
they both gave similar level of knockdown ,but the downstream effects (other proteins that were influenced)were not the same.
how is it possible? i compared them to the database-and they not supposed to knockdown any other gene. what can i do except try another sequence?
help me ...
It is not uncommon to find that two siRNA knock down a gene with similar efficiency but result in different phenotypic changes. All siRNAs can cause different degrees of off-target effects: targeting other sequences in a sequence specific manner such as acting as miRNA, interferon response and cytokine stimulation etc. In your case, probably you have to choose another siRNA.
Try to order the same siRNAs but with 2'O-Methylation at the 3' end of the passenger strand (the strand NOT complementary to your mRNA). Most RNA synthesis companies offer that modification and that is rather cheap. This decreases the chances for this strand being incorporated instead of the guide strand (the strand complementary to your mRNA) greatly. And the passenger strand can act at least as a microRNA like effector if it finds a seed region in another gene - thus causing off target effects.