BIOTECHNOLOGY 47


duplexes in vitro before moving to in vivo experiments.


In addition, choose more than one effective siRNA for each target to be tested, to rule out false positive results caused by off-target effects11


.


RNAi is a powerful tool for studying gene silencing and its effects. Advancements to the technology, such as IDT DsiRNAs, have led to even greater improvements in the potency of RNA interference.


Such tools will continue to provide the means to study the role specific genes play through the effects of silencing them.


Fig. 3. IDT uses proprietary synthesis platforms developed to provide the highest coupling efficiency.


moderate to low GC content (30–52 per cent) is typically a feature of functional siRNAs5


.


It is important to screen candidate siRNAs for homology to other targets and exclude those with significant complementarity5


.


BLAST is not a good tool for finding short 5- to 8-base domains of sequence identity between a candidate siRNA and other genes; the Smith-Waterman algorithm is recommended for siRNA homology screening instead5


. Programs such as


SSEARCH or JALIGNER are two free options for this type of analysis.


Like targeted effects, off-target effects (OTEs) are dose dependent. Terefore, it is important to establish dose-response profiles for all siRNAs; always use the lowest concentration of siRNA that will provide sufficient target knockdown. An additional measure to identify OTE bias is to ensure that at least two, and ideally three, independent siRNAs that target different sites of a specific target RNA transcript produce the same results5


.


siRNAs used in vivo show great potential as both research tools and as therapeutic agents11


review on the status of RNAi in therapeutics, see the recent article by Vaishnaw et al12


. Before you


begin RNAi studies in vivo, consider the following issues: site selection, siRNA design and chemistry, controls, route of administration, and use of a delivery vehicle11


.


To find the best candidates, it is important to validate siRNA


References 1


2 3 4


Definitions DsRNA -


Dicer - SiRNA-


RISC - Guide strand -


Jaime Sabel and Hans Packer are Scientific Writers at Integrated DNA Technologies (IDT), Coralville, IA, USA. http://eu.idtdna.com


double-stranded RNA.


an endoribonuclease that degrades long dsRNAs into small, effector molecules called siRNAs. small interfering RNA.


RNA-Induced Silencing Complex. Passenger strand—the sense strand of the siRNA; degraded by Dicer during RNAi processing.


the antisense strand of the siRNA; incorporated into RISC during RNAi processing.


Hannon GJ and Rossi JJ. (2004) Unlocking the potential of the human genome with RNA interference. Nature, 431(7006): 371–378.


Meister G and Tuschl T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature, 431(7006): 343–349.


Chendrimada TP, Gregory RI, et al. (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature, 436(7051): 740–744.


Whitehead KA, Langer R, and Anderson DG. (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov, 8(2): 129–138.


5 Peek AS and Behlke MA. (2007) Design of active small interfering RNAs. Curr Opin Mol Ter, 9(2): 110–118. 6


Kim DH, Behlke MA, et al. (2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol, 23(2): 222–226.


7 8


Rose SD, Kim DH, et al. (2005) Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res, 33(13): 4140–4156.


Amarzguioui M, Lundberg P, et al. (2006) Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat Protoc, 1(2): 508–517.


9 Behlke MA. (2008) Chemical modification of siRNAs for in vivo use. Oligonucleotides, 18(4): 305–319. 10


. For a


11 Behlke MA. (2006) Progress towards in vivo use of siRNAs. Mol Ter, 13(4): 644–670. 12 Vaishnaw AK, Gollob J, et al. (2010) A status report on RNAi therapeutics. Silence, 1(1): 14.


www.scientistlive.com


Strapps WR, Pickering V, et al. (2010) Te siRNA sequence and guide strand overhangs are determinants of in vivo duration of silencing. Nucleic Acids Res, 38: 4788–4797.


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