No change in mRNA or protein levels after RNAi in Trypanosoma brucei - (Jun/24/2005 )
Can somebody help! I am doing RNAi in monomorphic T.brucei strain 328:114. Ia observing a tremendous change in morphology and reduction in growth. Supprisengly I am not getting any reduction in mRNA or ptorein levels what could be the problem?
Have you transfected any control siRNA into the strain and did the strain show morphologic changes too? Are you using synthetic siRNA or shRNA? Morphologic changes and growth inhibition do not necessarily indicate target gene knockdown even the changes are sequence-specific (no changes for control siRNA) probably due to off-target effects.
I am using an inducible system pZJM vector. I have not transfected with control siRNA. Additionally the gene I am working on has no similarity with any other gene from the database, if siRNA fragements are targetting other genes because they might have similarity with some short sequences due to their small sizes, then at least I should also observe some reduction of target mRNA and protein.
If there is no control shRNA transfected, it is hard to tell if the changes are sequence specific. I don't know much about the organism you are using, but in mammals, siRNAs can also act on non-coding (or regulatory, promoter) sequence. Off-target effect has to be checked against those sequences not just mRNA sequences.
We have done a lot of RNAi in T. brucei in our lab but have never had much success with PZJM. You might find the p2T7Ti vector (Donelson lab) better. I have a few other suggestions that might help.
1) Check the integration site. Mistargeting of the vector has been reported (Motyka, Mol Biochem Parasitol. 134 p63, 2004). Repeating the transfection and testing several clones may reduce the chances of getting this kind of artefact.
2) Revertants which do not respond to the tetracycline can come through very quickly, especially when there is a severe toxic effect associated with the RNAi. Since the induced cells are likely to be sick, they will be more fragile than uninduced cells and more prone to loss during processing, so cells which still express your gene may be over-represented in your final protein or RNA prep. There may be a narrow time window where loss of expression is detectable, but the cells are healthy enough to use, so
3) Try using IFAT to detect your protein and confirm that the loss of expression is associated with the abnormal morphology. Again, the surviving cells may be those in which the RNAi is relatively inefficient, so the loss of protein may not be total.
4) How are you detecting your RNA? We found in some cases that semi-quantitative RT-PCR was more sensitive than a blot for detecting changes in expression. Also the nature of the probe can affect the result. I found sequences close to the target sequence but not overlapping were most reliable.
Hope this helps