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shRNA designed for AB1 gene also knockdown AB2 - (Nov/08/2012 )

I recently encoutered a mystery that my shRNA containing lentivirus knocked-down AB1 gene (a pseudoname) but also knocked down another homologue, AB2. How could this happen? I thought shRNA (designed by TRC) is very specific.

-Yen-Ling Chiu-

Have you checked the shRNA sequence to see if it targets AB2? You said the two genes are homologue, it is more often than not they share some sequence homology. the shRNA could have an effect on genes with which there is only partial homology.


This sort of effect is well documented. If a non-native sequence loads onto RISC, you will get off-target gene modulation.

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 2004 Aug 27;305(5688):1289-92. Epub 2004 Aug 5.

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.

Noise amidst the silence: off-target effects of siRNAs? Jackson AL, Linsley PS. Trends Genet. 2004 20(11):521-4

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.

-Jon Moulton-