Noise amidst the silence: off-target effects of siRNAs?
Aimee L. Jackson and Peter S. Linsley
Rosetta Inpharmatics LLC*, 401 Terry Avenue N., Seattle, WA 98109, USA
RNA interference (RNAi), mediated by short interfering
RNAs (siRNAs), is widely used to silence gene expres-
sion and to define gene function in mammalian cells.
Initially, this gene silencing via transcript degradation
was believed to be exquisitely specific, requiring near-
identity between the siRNA and the target mRNA.
However, several recent reports have suggested that
non-specific effects can be induced by siRNAs, both at
the level of mRNA and protein. These findings suggest
that siRNAs can regulate the expression of unintended
targets, and argue for further experiments on the mech-
anism and extent of off-target gene regulation(s).
In the meantime, caution is warranted in interpret-
ing gene function and phenotypes resulting from
The current challenge facing the use of RNA interference
(RNAi) to define gene function is the need to combine high
specificity with high efficacy. Ineffective siRNAs can lead
to false negative conclusions, whereas non-specific siRNAs
can produce false-positive conclusions regarding the role
of the target gene in functional assays. Efforts have been
made to improve the design of siRNAs for increased
efficacy, including optimizing the base composition profiles
to affect the relative ease of unwinding the RNA duplex.
These design efforts have converged on the theme of
directing the active, antisense, strand of the siRNA into an
RNA-induced silencing complex (RISC), and are described
in detail elsewhere [1–4]. These efforts have led to
considerable improvement in the rational design of
siRNAs. The incorporation of these design criteria is
expected to enhance the probability of gene silencing with
high efficacy (end-point silencing) and potency (lowest
concentration of siRNA that produces full silencing).
However, the question of siRNA specificity lingers.
Ideally, siRNAs would be absolutely specific, regulating
only the target gene of interest (Figure 1a). However, a
growing body of evidence suggests that this is not
necessarily the case. siRNAs were first reported to affect
unintended mRNA transcripts that are partially comp-
lementary in sequence  (Figure 1c). More recently,
siRNAs have been reported to affect translation of
unintended transcripts containing partial complementar-
ity [6,7], and to induce the non-specific interferon response
[8,9] (Figure 1b).
The identification and functions of small non-coding
RNAs, such as siRNAs and microRNAs (miRNAs; Box 1),
have been increasing exponentially, revealing numerous
ways in which these RNAs can regulate gene expression.
These include degradation of mRNA, inhibition of trans-
lation and regulation of heterochromatin. The rules
distinguishing the function of siRNAs from miRNAs are
not well understood, and the distinction between these
RNAs might be even less than we appreciate. siRNAs
might be able to function as miRNAs and vice versa. These
findings suggest that siRNAs might have multiple effects
on gene expression. Understanding these ‘off-target’
effects is crucial for accurate interpretation of gene
function by RNAi and will enable improved design of
more specific siRNAs.
TRENDS in Genetics
Figure 1. Types of short interfering RNAs (siRNA)-induced gene silencing responses. Schematics of potential microarray gene expression signatures induced by siRNAs
are shown. Hypothetical regulated genes are arranged on the x-axis, whereas different siRNAs (experiments) are arrayed on the y-axis. Different siRNAs are grouped
together by their intended targets, with three siRNAs per gene. Genes that are upregulated in siRNA-transfected cells are shown in red, genes that are downregulated
in siRNA-transfected cells are shown in green. (a) The target-specific response. Multiple siRNAs for the same target gene produce identical expression signatures and these
signatures are different from siRNAs for a different target gene. (b) The interferon response. Multiple siRNAs for the same target gene produce identical expression
signatures, however, these are identical to siRNAs targeting a different gene. These are non-specific off-target effects. (c) The siRNA-specific response. Different siRNAs for
the same target gene produce unique expression signatures. A subset of the signature might be overlapping and indicate on-target gene silencing; however, in some cases,
the overall signature might be dominated by off-target effects.
Corresponding author: Aimee L. Jackson (firstname.lastname@example.org).
* Rosetta Inpharmatics LLC is a wholly-owned subsidiary of Merck & Co.
Available online 11 September 2004
TRENDS in GeneticsVol.20 No.11 November 2004