RNA References

General Reviews

Dykxhoorn, D.M. et al. (2003). Killing the messenger: Short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4: 457-467. Provides a broad review of RNAi in mammalian systems.

Wasi, S. (2003). RNA interference: The next genetics revolution? from Horizon Symposia; Understanding the RNAissance. Nature Pub. Group, pp. 1-4. Discusses potential therapeutic applications of RNAi.

Zamore, P.D. (2002). Ancient pathways programmed by small RNAs. Science 296: 1265-1269. Provides a broad review of RNAi.

siRNA

Caplen, N.J. et al. (2001). Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. U S A. 98: 9742-9747. This paper establishes the use of synthetic siRNA to silence genes in mammalian cells.

Elbashir, S.M. et al. (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15: 188-200. The authors demonstrate that synthetic siRNA can initiate RNAi in Drosophila embryo extracts.

Elbashir. S.M. et al. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-498. This paper establishes the use of synthetic siRNA to silence genes in mammalian cells.

Elbashir, S.M. et al. (2001). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20: 6877-6888. The length, overhangs, and sequences of synthetic siRNA are tested for their effects on silencing efficacy and the position of the mRNA cleavage site.

Hamilton, A.J. and Baulcombe, D.C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286: 950-952. The first recognition that siRNA are produced in the cell and are associated with gene silencing.

siRNA Target Selection

Elbashir, S.M. et al. (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26: 199-213. Summarizes the early rules developed in Tuschl's lab for siRNA design.

Khvorova, A. et al. (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell 115: 209-216. Surveys hundreds of functional and non-functional siRNA to demonstrate the importance of duplex stability in determining the efficiency of mRNA cleavage.

Schwarz, D.S. et al. (2003). Asymmetry in the assembly of the RNAi enzyme complex. Cell 115: 199-208. The relative stability of each end of the siRNA duplex is shown to be critical in determining which strand is loaded into the RISC. For knockdown to occur, the antisense strand must be loaded into the RISC.

RISC

Hammond, S.M. et al. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293-296. Describes the biochemical isolation of the RISC and the identification of a small RNA component.

Hammond, S.M. et al. (2001). Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293: 1146-1150. A member of the extensive Argonaute family of proteins genetically linked to gene silencing is identified as a component of the RISC.

Martinez, J. and Tuschl, T. (2004). RISC is a 5' phophomonoester-producing RNA endonuclease. Genes Dev. 18: 975-980. Describes the biochemical activity of the RISC nuclease.

Schwarz, D. (2004). The RNA-induced silencing complex is a Mg(2+)-dependent endonuclease. Curr. Biol. 14: 787-791. Describes the biochemical activity of the RISC nuclease.

Dicer

Ueda, K. et. al. (2004) Proteomic identification of Bcl-2-associated Athanogene 2 as a novel MAPK-activated protein kinase 2 substrate. J. Biol. Chem. 279: 41815-41821. Researchers use BLOCK-iT™ Dicer Transfection Kit for analysis presented in this paper.

Ambros, V. (2001). Development. Dicing up RNAs. Science 293: 811-813. Reviews the role of Dicer in the RNAi and miRNA pathways.

Bernstein, E. et al. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363-366. Identifies Dicer as the RNaseIII responsible for generation of siRNA in Drosophila extracts.

Myers, J.W. et al. (2003). Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat. Biotechnol. 21: 324-328. This paper demonstrates that siRNA created in vitro by recombinant human Dicer are effective for RNAi in mammalian cells.

Zhang, H. et al. (2002). Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21: 5875-5885. The authors explore the mechanism by which recombinant human Dicer cleaves dsRNA in vitro.

shRNA Plasmid Vectors

Brummelkamp, T.R. et al. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553. The human H1 promoter is used to express shRNA from a plasmid vector in mammalian cells.

Czauderna, F. et al. (2003). Inducible shRNA expression for application in a prostate cancer mouse model. Nuc. Acids Res. 31: E127. The authors use Invitrogen's T-REx ™ system and a tetracycline-responsive operator (tetO) site in the U6 promoter to regulate shRNA expression.

Paddison, P.J. et al. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16: 948-958. The human U6 promoter is used to express shRNA from a plasmid vector in mammalian cells.

Tuschl, T. (2002). Expanding small RNA interference. Nat. Biotechnol. 20: 446-448. News and Views summary of early shRNA vector papers.

Viral Vectors

Arts, G.J. et al. (2003). Adenoviral vectors expressing siRNAs for discovery and validation of gene function. Genome Res. 13: 2325-2332. Demonstrates adenovirus-based shRNA expression in a variety of cell types, including primary cells.

Matta, H. et al. (2003). Use of lentiviral vectors for delivery of small interfering RNA. Cancer Biol. Ther. 2: 206-210. The authors use Invitrogen's pLenti6 backbone to express an shRNA cassette.

Tiscornia, G. et al. (2003). A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc. Natl. Acad. Sci. USA 100: 1844-1848. This paper demonstrates the utility of lentiviral vectors for delivery of shRNA to cells and mice.

miRNA

Bartel, D.P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116: 281-297. This comprehensive review covers miRNAs from transcription to targets.

Lagos-Quintana, M. et al. (2001). Identification of novel genes coding for small expressed RNAs. Science 294: 853-858. This paper identifies the abundant number of microRNA in Drosophila and humans.

Zeng, Y. et al. (2003). MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc. Natl. Acad. Sci. USA 100: 9779-9784. This paper demonstrates that siRNA and miRNA can be functionally interchangeable.

Off-Target and Non-Specific Effects

Off-Target Effects
Jackson, A.L. et al. (2003). Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21: 635-637. Microarray data reveal off-targets effects from siRNA.

Persengiev, S.P. et al. (2004). Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10: 12-18. A large number of genes are shown to be non-specifically stimulated and repressed by synthetic siRNA.

Saxena, S. et al. (2003). Small RNAs with imperfect match to endogenous mRNA repress translation: Implications for off-target activity of siRNA in mammalian cells. J. Biol. Chem. 278: 44312-44319. Discusses implications of off-target activation of the translational repression pathway of miRNA by siRNA.

Non-Specific Effects – Induction of the Interferon Pathway
Bridge, A.J. et al. (2003). Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet. 34: 263-264. shRNA expressed from a plasmid vector can induce the interferon pathway.

Moss, E.G. and Taylor, J.M. (2003). Small-interfering RNAs in the radar of the interferon system. Nat. Cell Biol. 5: 771-772. This commentary summarizes the findings of the Sledz et al. paper.

Pebernard, S. and Iggo, R.D. (2004). Determinants of interferon-stimulated gene induction by RNAi vectors. Differentiation 72: 103-111. An extension of the Bridge et al. paper showing that interferon stimulation from U6-based vectors occurs when adenosines are present in the -1/+1 positions.

Note: pENTR™/U6 constructs designed at https://rnaidesigner.lifetechnologies.com do not have this motif.

Sledz, C.A. et al. (2003). Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5: 834-839. Synthetic siRNA are capable of activating the interferon pathway.

Controls

Editors (2003). Whither RNAi? Nat. Cell Biol. 5: 489-490. Provides recommendations for the necessary controls to include in RNAi experiments.

Lassus, P. et al. (2002). Confirming specificity of RNAi in mammalian cells. Sci. STKE 2002(147): PL13. Discusses the importance of using a “rescue” vector control to confirm specificity of the knockdown phenotype.