different siRNA for the same gene cause opposite effect - (Oct/20/2005 )
I generated five different siRNA oligos against the same mRNA, When I transfect them I observe some oposed secondary effects with some of them, working all of them to knock down the gene of my interest. How could I know which one causes non specific secondary effects?
Any help would be much apreciated.
hi
you must check the interferon induction (not suppose to occurs)
at mRNA level (chack OAS1 one)
at protein level : STAT 1 becomes phopshorylated
second you may check for similar proteins as the one you want to knock down and check if they remain unafected at both mRNA and protein level.
fred
I would say that the inconsistency is probably due to observing variations. What method do you use to determine the effect? You have to repeat the experiment at least three times to see if you can reproduce the result.
The effect I observe, is at protein level. By means of westernblot, I can see oposed protein variation. The amount of protein loaded in each case is always the same as well as all the processing of the samples for each oligo. I have repeated this experiment four times obtaining always the same effects.
In that case, the induction effect is probably true. There are two possibilities, interferon response or induction at transcriptional level. Could you send me your siRNA sequence and the gene name so I can have a look?
I thought Interferon response was only possible using RNAi vector, but not using oligos. Is then that possible?
Excuse me, I apreciate your help, and I don't doubt about your intentions to help, but I am not authorizied to give you the information you ask me.
Thanks
Never mind.
Yes, there are a few papers showing synthetic siRNA causes interferon response although I don't believe it.
Here is one paper http://www.ncbi.nlm.nih.gov/entrez/query.f...2087&query_hl=3