confusion about off-target effect - (Oct/24/2008 )
I'm working in the cardiovascular field, where it has become very hip to use siRNA as a tool to study protein function. Unfortunately, I got the impression that very few scientists use the proper controls. Most of the published data were obtained using only one siRNA sequence for knock down without any chemical modifications. And that data is published in not so bad ranking journals.
After all I've read about off-targeting I really don't understand how this is possible. Do you?
As an example, I'd like to show you one paper published in the Journal of Molecular and Cellular Cardiology in May 2008:
Here, a knockdown of a channel subunit (KChIP2) was performed via siRNA. They used one single sequence and performed a Blast (!!!!) search to check for matches with other genes. At least they used two different control sequences: one commercially available and one having the same sequence as the siRNA with a single base pair mismatch at position 8. And - surprise! They found that the mRNA of another channel (Navbeta1) was being downregulated! For them it's a strong hint towards a functional coupling.
Okay, to calm down: they also silenced Navbeta1 with another siRNA supposed to be specific for it and saw the same effect vice versa.
But still: in the year 2008 how is it possible, that somebody writes: a Blast search was performed to reveal matches with other genes?
Am I crazy or are they?????
You're not crazy.
Widespread changes in protein synthesis induced by microRNAs. Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Nature. 2008 Jul 30. [Epub ahead of print]
Comparison of siRNA-induced off-target RNA and protein effects. Aleman LM, Doench J, Sharp PA. RNA. 2007 Jan 19; [Epub ahead of print]
Retraction of synapses and dendritic spines induced by off-target effects of RNA interference. Alvarez VA, Ridenour DA, Sabatini BL. J Neurosci. 2006 Jul 26;26(30):7820-5.
3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, Baskerville S, Maksimova E, Robinson K, Karpilow J, Marshall WS, Khvorova A. Nat Methods. 2006 Mar;3(3):199-204.
Nonspecific, concentration-dependant stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). Persengiev SP, Zhu X and Green M. RNA 2004; 10:12-18.
Transcriptional gene silencing by short interfering RNAs. Kawasaki H, Taira K. Curr Opin Molec Therap. 2005 7(2):125-31.
Small interfering RNA-induced transcriptional gene silencing in human cells. Morris KV, Chan SWL, Jacobson SE, Looney DJ. Science 305(5688):1289-92.
A rapid and sensitive assay for quantification of siRNA efficiency and specificity. Smart N, Scambler PJ, Riley PR. Biol Proced Online. 2005;7:1-7. Epub 2005 Jan 24.
Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7. Epub 2004 Feb 09.
Expression profiling reveals off-target gene regulation by RNAi. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G, Linsley PS. Nat Biotechnol. 2003 Jun;21(6):635-637.
Small RNAs with Imperfect Match to Endogenous mRNA Repress Translation: Implications for off-target activity of small inhibitory RNA in mammalian cells. Saxena S, Jonsson ZO, Dutta A. J Biol Chem. 2003 Nov 7;278(45):44312-44319.
Color me confused - what is the problem with screening for off-targets with BLAST?
Is it because of the seed sequence being responsible for off-targets, or just that BLAST isn't the right tool here...
The seed sequence is pretty short. There are many complementary regions in a transcriptome to a recognition sequence of eight bases length.