Recommended Reading in siRNA Research
Frequently cited siRNA-related articles from peer-reviewed journals and scientific texts.
GETTING STARTED
The following articles describe important insights into the history and uses of RNAi.
RNAi in review - Many excellent reviews on RNAi can be found in the literature. Here are a few favorites.
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Zamore, P. D. (2001) RNA interference: listening to the sound of silence. Nat Struct Biol 8, 746–750.
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Tuschl, T. (2001). RNA Interference and Small Interfering RNAs. Chembiochem Europ J Chem Biol 2, 239245.
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Hannon, G. J. (2002). RNA interference. Nature 418, 244-251. A thorough review of the underlying biology
and mechanics of RNA interference.
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Agrawal, N., Dasaradhi, P. V. N., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., and Mukherjee, S. K. (2003).
RNA Interference: Biology, Mechanism, and Applications. Microbiol Mol Biol Rev 67, 657-685. A
comprehensive review of the biology and proposed mechanism of RNA interference with over 200 references.
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Bonetta, L. (2004) RNAi: Silencing never sounded better. Nature Methods. 1:79-86.
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Gilmore, I., S. Fox, A. Hollins, M. Sohail and S. Akhtar. (2004) The design and exogenous delivery of siRNA
for post-transcriptional gene silencing. J Drug Target. 12:315-40.
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Sandy, P. V., Andrea. Jacks, Tyler. (2005) Mammalian RNAi: a practical guide. Bio Techniques. 39:1-10.
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Dykxhoorn, D. and J. Lieberman. (2005) The silent revolution: RNA interference as basic biology, research
tool, and therapeutic. Annu Rev Med. 56:401-23.
MILESTONES IN siRNA RESEARCH
Early siRNA Research across the phyla (Petunia, Neurospora, and C. elegans)
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Napoli, C., Lemieux, C., and Jorgensen, R. (1990). Introduction of a Chimeric Chalcone Synthase Gene into
Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2, 279-289.
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Romano, N., and Macino, G. (1992). Quelling: transient inactivation of gene expression in Neurospora crassa by
transformation with homologous sequences. Mol Microbiol 6, 3343-3353.
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Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998). Potent and specific
genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811. The first report
showing that double-stranded RNA (dsRNA)—comprising both sense and antisense strands—is far more potent
than the individual antisense or sense strands alone in silencing gene targets.
Demonstrations of siRNA knockdown in mammalian cells
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Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001). Duplexes of 21nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498.
In this landmark publication, these authors demonstrate the utility of chemically synthesized siRNAs for targeted
gene knockdown in vertebrates, effectively bypassing the interferon response.
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Caplen, N. J., Parrish, S., Imani, F., Fire, A., and Morgan, R. A. (2001). Specific inhibition of gene expression by
small double-stranded RNAs in invertebrate and vertebrate systems. PNAS 98, 9742-9747. This work provided
independent support for the ability to achieve siRNA-mediated silencing in vertebrates cell culture.
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Recommended Reading in siRNA Research
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Bantounas, I., L. A. Phylactou and J. B. Uney. (2004) RNA interference and the use of small interfering RNA to
study gene function in mammalian systems. J. Mol. Endocrinol. 33:545-557.
Demonstration of siRNA silencing in vertebrates
Proof-of-principle studies demonstrating the utility of siRNA or shRNA-mediated silencing in animal models.
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McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., and Kay, M. A. (2002). Gene
expression: RNA interference in adult mice. Nature 418, 38-39.
This report demonstrates the utility of siRNAs and shRNAs as potential therapeutic agents whether delivered as
chemical entities or expressed from vectors.
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Lewis, D. L., Hagstrom, J. E., Loomis, A. G., Wolff, J. A., and Herweijer, H. (2002). Efficient delivery of siRNA for
inhibition of gene expression in postnatal mice. Nat Genet 32, 107.
This paper describes efficient delivery of siRNAs via hydrodynamic tail injections and subsequent silencing of coinjected reporter plasmids in a dose dependent manner.
Demonstration of siRNA silencing in Plants, Pathogens, and Virus
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Susi, P., M. Hohkuri, T. Wahlroos and N. J. Kilby. (2004) Characteristics of RNA silencing in plants: similarities
and differences across kingdoms. Plant Mol Biol. 54:157-74.
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Cottrell, T. and T. Doering. (2003) Silence of the strands: RNA interference in eukaryotic pathogens. Trends
Microbiol. 11:37-43.
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Tan, F. and J. Yin. (2004) RNAi, a new therapeutic strategy against viral infection. Cell Res. 14:460-6.
CONSIDERATIONS AND ELEMENTS OF siRNA DESIGN
Designing your experiment: A list of references describing key parameters of siRNA design.
Recommendations for experimental setup
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Editorial (2003) Whither RNAi? In Nat Cell Biol, pp. 489-490.
A concise editorial proposing adoption of good experimental practices for RNAi research by academia and
industry. These include the use of appropriate controls, biological replicates, confirmation of knockdown with
multiple, independent RNAi effectors, and confirmation via distinct detection methods (mRNA levels, protein
levels, and phenotype).
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Huppi, K., Martin, S. E., and Caplen N.J. (2004) Defining and Assaying RNAi in Mammalian Cells. Mol Cell
17, 1-10.
A review that reiterates good experimental practice with emphasis on attributes associated with expressed
shRNA (i.e., confirming sequence fidelity, expression levels, regulation of expression, consideration of the mRNA
target and its biological context – turnover rates, redundancy and feedback regulation)
Aspects of siRNA design
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Schwarz, D. S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P. D. (2003). Unexpected Asymmetry in
the assembly of the RNAi Enzyme Complex. Cell 115, 199-208.
Biochemical studies that identified a bias or asymmetry in functional siRNAs that was not present in nonfunctional duplexes.
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Khvorova, A., Reynolds, A., and Jayasena, S. (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell
115, 209-216.
The authors compare closely related classes of small regulatory RNAs, microRNAs, and siRNAs to identify key
attributes for efficient processing by the RISC.
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Reynolds, A., Leake, D., Scaringe, S., Marshall, W. S., Boese, Q., and Khvorova, A. (2004). Rational siRNA
design for RNA interference. Nature Biotechnology 22(3), 326 – 330.
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Recommended Reading in siRNA Research
This work represents the first rigorous, proof-of-concept study in a peer-reviewed journal documenting the
feasibility of rational siRNA design. The rational design detailed in the publication was extended to include many
additional parameters now used in Dharmacon's proprietary SMARTselectionTM technology platform.
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Kim, D-H, Behlke, M. A., Rose, S.D., Chang, M-S., Choi S., and Rossi, J.J. (2004) Synthetic dsRNA Dicer
substrates enhance RNAi potency and efficacy. Nature Biotechnology Advanced Online Publication January
2004.
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Despina Siolas, D., Lerner, C., Burchard, J., Ge, W., Linsley, P. S., Paddison, P.J. Hannon, G.J. and Cleary,
M.A. (2004) Synthetic shRNAs as potent RNAi triggers. Nature Biotechnology Advanced Online Publication
January 2004.
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Saetrom, P. and O. Snove. (2004) A comparison of siRNA efficacy predictors. Biochem Biophys Res
Commun. 321:247-53.
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Amarzguioui, M. and H. Prydz. (2004) An algorithm for selection of functional siRNA sequences. Biochem
Biophys Res Commun. 316:1050-8.
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Ui-Tei, K., Y. Naito, F. Takahashi, T. Haraguchi, H. Ohki-Hamazaki, A. Juni, R. Ueda and K. Saigo. (2004)
Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.
Nucleic Acids Res. 32:936-948.
Experimental considerations: countering potential off-target effects
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Chi J-T, Chang H. Y., Wang N. N., Chang D. S., Dunphy N., Brown P. O. (2003) Genome-wide view of gene
silencing by small interfering RNAs. PNAS 100, 6343–6346.
These authors used conventionally designed siRNAs and microarray gene profiling to assess potential off-target
effects but observed no consistent silencing pattern for unintended targets.
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Jackson A. L., Bartz S. R., Schelter J., Kobayashi S. V., Burchard J., Mao M., Li B., Cavet G., Linsley P. S.
(2003) Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology 21, 635–637.
Conventionally designed siRNAs demonstrated off-target, homology-directed effects by microarray gene
profiling, however, in an on-going collaboration with Dharmacon, sense-strand off-target effects were diminished
with the application of ON-TARGET™ modifications to the cognate siRNAs (unpublished work).
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Semizarov D., Frost L., Sarthy A., Kroeger P., Halbert D. N., Fesik S. W. Specificity of short interfering RNA
determined through gene expression signatures. PNAS 100, 6347–6352.
Observations of siRNA induced off-target effects were diminished by reducing concentrations. Of note, siRNAs
were selected by the application of additional thermodynamic criteria to conventional design further highlighting
the utility of rational, SMARTselection design.
SELECTED REFERENCES DOCUMENTING IN VIVO STUDIES
For additional information please see our flier “in vivo RNA interference publications” at www.biosyn.com
Soutschek, J., et al. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified
siRNAs. Nature. 432:173-8.
Watanabe, H., H. Saito, P. Rychahou, T. Uchida and B. M. Evers. (2005) Aging is Associated with Decreased
Pancreatic Acinar Cell Regeneration and Phosphatidylinositol 3-kinase/Akt Activation. Gastroenterology.
128:1391-404.
612 E. Main Street • Lewisville, TX 75057 • www.biosyn.com
Phone: (972) 420-8505 • Toll Free: (800) 227-0627 • Fax (972) 420-0442
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