![]() RNA interference ( RNAi ) & basic and essential siRNA publication (3) siRNA & quantitative real-time RT-PCR (1) siRNA & quantitative real-time RT-PCR (2) siRNA & quantitative real-time RT-PCR (4)
RNA interference: unraveling a mystery Mary K Montgomery NATURE STRUCTURAL & MOLECULAR BIOLOGY (2006) VOLUME 13 NUMBER 12 1039 Andrew Fire and Craig Mello have won the Nobel Prize in Medicine or Physiology for their discovery of RNA interference. Mary K. Montgomery, then a postdoc in the Fire laboratory, participated in some of the key experiments. siRNAdb: a database of siRNA sequences Alistair M. Chalk, Richard E. Warfinge, Patrick Georgii-Hemming and Erik L. L. Sonnhammer Nucleic Acids Research, 2005, Vol. 33, Database issue Center for Genomics and Bioinformatics, Karolinska Institutet, Berzelius va¨g 35, S-171 77 Stockholm, Sweden The database is available at http://siRNA.cgb.ki.se Short interferingRNAs
(siRNAs) are a popular method for gene-knockdown, acting by degrading
the target mRNA. Before performing experiments it is invaluable to
locate and evaluate previous knockdown experiments for the gene of
interest. The siRNA databaseprovides a gene-centric view
of siRNA experimental data, including siRNAs of known efficacy and
siRNAs predicted to be of high efficacy by a combination of methods.
Linked to these sequences is information such as siRNA thermodynamic
properties and the potential for sequence-specific
off-target effects. The database enables the user to evaluate an
siRNA’s potential for inhibition and non-specific effects.
Agenome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Georg Dietzl, Doris Chen, Frank Schnorrer, Kuan-Chung Su, Yulia Barinova, Michaela Fellner, Beate Gasser, Kaolin Kinsey, Silvia Oppel, Susanne Scheiblauer, Africa Couto, Vincent Marra, Krystyna Keleman & Barry J. Dickson Nature (2007) Vol 448 The library is available at http://www.vdrc.at Forward genetic screens
in model organisms have provided important insights into numerous
aspects of development, physiology and pathology. With the availability
of complete genome sequences and the introduction of RNA-mediated gene
interference (RNAi), systematic reverse genetic screens are now also
possible. Until now, such genome-wide RNAi screens have mostly been
restricted to cultured cells and ubiquitous gene inactivation in
Caenorhabditis elegans. This powerful approach has not yet been applied
in a tissue-specific manner. Here we report the generation and
validation of a genome-wide library of Drosophila melanogaster RNAi
transgenes, enabling the conditional inactivation of gene function in
specific tissues of the intact organism. Our RNAi transgenes consist of
short gene fragments cloned as inverted repeats andexpressed
using the binary GAL4/UAS system. We generated 22,270 transgenic lines,
covering 88% of the predicted protein-coding genes in
the Drosophila genome. Molecular and phenotypic assays indicate that
the majority of these transgenes are functional. Our transgenic RNAi
library thus opens up the prospect of systematically analysing gene
functions in any tissue and at any stage of the Drosophila lifespan.
dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference. Yuki Naito, Tomoyuki Yamada, Takahiro Matsumiya, Kumiko Ui-Tei1, Kaoru Saigo1 and Shinichi Morishita Nucleic Acids Research, 2005, Vol. 33, Web Server issue The software is available at http://dsCheck.RNAi.jp/ Off-target effects are
one of the most serious problems in RNA interference (RNAi). Here, we
present dsCheck (http://dsCheck.RNAi.jp/), web-based online software
for estimating off-target effects caused by the long double-stranded
RNA (dsRNA) used in RNAi studies. In the biochemical process of RNAi,
the long dsRNA is cleaved by Dicer into shortinterferingRNA (siRNA)
cocktails. The software simulates this process and investigates
individual 19 nt substrings of the longdsRNA. Subsequently, the
software promptly enumerates a list of potential off-target gene
candidates based on the order of off-target effects using its novel
algorithm, which significantly improves both the efficiency and the
sensitivity of the homology search. The website not only provides a
rigorous off-target search to verify previously designed dsRNA
sequences but also presents ‘offtarget minimized’ dsRNA design, which
is essential for reliable experiments in RNAi-based functional genomics.
RNAi Codex: a portal/database for short-hairpin RNA (shRNA) gene-silencing constructs A. Olson, N. Sheth, J. S. Lee, G. Hannon and R. Sachidanandam* Nucle ic Acids Research, 2006, Vol. 34, Databas e issue The GeneSeer service is available at http://geneseer.cshl.org Use of RNA interference
(RNAi) in forward genetic screens is proliferating. Currently,
short-interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs) are
being used to silence genes to tease out functional information. It is
becoming easier to harness RNAi to silence specific genes, owing to the
development of libraries of readymade shRNA and siRNA genesilencing
constructs by using a variety of sources. RNAi Codex, which consists of
a database of shRNA related information and an associated website, has
been developed as a portal for publicly available shRNA resources and
is accessible at http://codex.cshl.org. RNAi Codex currently holds data
from the Hannon–Elledge shRNA library and allows the use of
biologist-friendly gene names to access information on shRNA constructs
that can silence the gene of interest. It is designed to hold
usercontributed annotations and publications for each construct, as and
when such data become available. We will describe features of RNAi
Codex and explain the use of the tool.
A universal RNAi-based logic evaluator that operates in mammalian cells. Keller Rinaudo, Leonidas Bleris, Rohan Maddamsetti, Sairam Subramanian, Ron Weiss & Yaakov Benenson NATURE BIOTECHNOLOGY (23007) VOLUME 25 NUMBER 7 795 Molecular automata that
combine sensing, computation and actuation enable programmable
manipulation of biological systems. We use RNA interference (RNAi) in
human kidney cells to construct a molecular computing core that
implements general Boolean logic to make decisions based on endogenous
molecular inputs. The state of an endogenous input is encoded by the
presence or absence of ‘mediator’
small interfering RNAs
(siRNAs). The encoding rules, combined with a specific arrangement of
the siRNA targets in a synthetic gene network, allow direct evaluation
of any Boolean expression in standard forms using siRNAs and indirect
evaluation using endogenous inputs. We demonstrate direct evaluation of
expressions with up to five logic variables. Implementation of the
encoding rules through sensory up- and down-regulatory links between
the inputs and siRNA mediators will allow arbitrary Boolean
decision-making using these inputs.
A computational study of off-target effects of RNA interference. Shibin Qiu, Coen M. Adema1 and Terran Lane* Department of Computer Science and 1Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA 1834–1847 Nucleic Acids Research, 2005, Vol. 33, No. 6 <>RNA interference
(RNAi)
is an intracellular mechanism for post-transcriptional gene silencing
that is frequently used to study gene function. RNAi is initiated by
short interfering RNA (siRNA) of 21 nt in length, either generated
from the double-stranded RNA(dsRNA)byusing theenzymeDicer or introduced
experimentally. Following association with an RNAi silencing complex,
siRNA targets mRNA transcripts that
have
sequence
identity for destruction. A phenotype resulting from this knockdown of
expression may inform about the function of the targeted gene. However,
‘off-target effects’ compromise the specificity of RNAi if sequence
identity between siRNA and random mRNA transcripts causes RNAi to
knockdown expression of non-targeted genes. The complete off-target
effects must be investigated systematically on each gene in a genome by
adjusting a group of parameters, which is too expensive to conduct
experimentally and motivates a study in silico. This computational
study examined the potential for off-target effects of RNAi, employing
the genome and transcriptome sequence data of Homo sapiens,
Caenorhabditis elegans and Schizosaccharomyces pombe. The chance for
RNAi off-target effects proved considerable, ranging from 5 to 80% for
each of the organisms, when using as parameter the exact identity
between any possible
siRNA sequences (arbitrary length ranging from 17 to 28 nt) derived
from a dsRNA (range 100–400 nt) representing the coding sequences of
target genes and all other siRNAs within the genome. Remarkably,
high-sequence specificity and low probability for
off-target reactivity were optimally balanced for siRNA of 21 nt, the
length observed mostly in vivo. The chance for off-target RNAi
increased (although not always significantly) with greater length of
the initial dsRNA sequence, inclusion into the analysis of available
untranslated region sequences and allowing for mismatches between siRNA
and target sequences. siRNA sequences from within
100 nt of
the 50 termini of coding sequences had low chances for off-target
reactivity. This may be owing to coding constraints for signal
peptide-encoding regions of genes relative to regions that encode for
mature proteins. Off-target distribution varied along the chromosomes
of C.elegans,apparentlyowingto theuseofmoreunique sequences in
gene-dense regions. Finally, biological and thermodynamical descriptors
of effective siRNA reduced the number of potential siRNAs compared with
those identified by sequence identity alone, but off-target RNAi
remained likely, with an offtarget error rate of 10%. These results
also suggest a direction for future in vivo studies that could both
help in calibrating true off-target rates in living organisms and also
in contributing evidence toward the debate of
whether siRNA efficacy is correlated with,
or independent of, the target molecule. In summary, off-target effects
present a real but not prohibitive concern that should be considered
for RNAi experiments.
Analysis of small RNAs with the Agilent 2100 Bioanalyzer NATURE METHODS AUGUST 2006 The Agilent 2100
Bioanalyzer offers advantages of sensitivity and accuracy for
performing RNA separation, detection and quantitation, coupled with a
rapid, automated system. Here we demonstrate the performance of the
Agilent 2100 Bioanalyzer compared with standard techniques for RNA
separation, detection and quantitation.
RNA in control Benjamin J. Blencowe and May Khanna NATURE (2007) Vol 447 In bacteria, some messenger RNAs can sense the need for their protein product and accordingly regulate expression of their own genes. A similar type of RNA regulation has now been revealed in higher organisms. Introduction of silencing-inducing transgenes does not affect expression of known transcripts Thierry Aelbrecht, Marnik Vuylsteke, Melanie Bauwens, Helena Van Houdt, Ann Depicker FEBS Letters 580 (2006) 4154–4159 While the RNA
interference (RNAi) mechanism has only been discovered a decade ago,
RNAi is now often used to study gene function by sequence-specific
knockdown of gene expression. However, it is still unknown whether
introduction of silencing-inducing transgenes alters the transcriptome.
To address this question, genome-wide transcriptional changes in
silenced and non-silenced backgrounds were monitored through microarray
analysis. No significant transcriptional changes were detected when
compared to the non-silenced control. This result was confirmed by
real-time polymerase chain reaction analysis of genes known to be
involved in RNA silencing. In conclusion,
introduction of
silencing-inducing constructs does not affect expression of known
transcripts in other genes than in those homologous to the targeted
ones. Consequently, when gene function is studied by RNAi, the
transcriptional changes detected will specifically be the result of
knockout of the gene of interest, at least for the genes present on the
array used in our study.
PCR-based generation of shRNA libraries from cDNAs BMC Biotechnology 2006, 6:28 Cheng Du, Baosheng Ge, Zhongfeng Liu, Kai Fu, Wing C Chan, Timothy W McKeithan Background: The use of
small interfering RNAs (siRNAs) to silence target gene expression has
greatly facilitated mammalian genetic analysis by generating
loss-offunction mutants. In recent years, high-throughput, genome-wide
screening of siRNA libraries has emerged as a viable approach. Two
different methods have been used to generate short hairpin RNA (shRNA)
libraries; one is to use chemically synthesized oligonucleotides, and
the other is to convert complementary DNAs (cDNAs) into shRNA cassettes
enzymatically. The high cost of chemical synthesis and the low
efficiency of the enzymatic approach have hampered the widespread use
of screening with shRNA
libraries. Results: We
report here an improved method for constructing genome-wide shRNA
libraries enzymatically. The method includes steps of cDNA
fragmentation and endonuclease MmeI digestion to generate 19-bp
fragments, capping the 19-bp cDNA fragments with a hairpin
oligonucleotide, and amplification of the hairpin structures by PCR.
The PCR step converts hairpins into double-stranded DNAs that contain
head-tohead cDNA fragments that can be cloned into a vector downstream
of a Pol III promoter. Conclusion: This method can readily be used to
generate shRNA libraries from a small amount of mRNA and thus can be
used to create cell- or tissue-specific libraries.
High-throughput RNAi screening in cultured cells: a user’s guide Christophe J. Echeverri and Norbert Perrimon NATURE REVIEWS GENETICS VOLUME 7 MAY 2006 373 Abstract | RNA
interference has re-energized the field of functional genomics by
enabling genome-scale loss-of-function screens in cultured cells.
Looking back on the lessons that have been learned from the first wave
of technology developments and applications in this exciting field, we
provide both a user’s guide for newcomers to the field and a detailed
examination of some more complex issues, particularly concerning
optimization and quality control, for more advanced users. From a
discussion of cell lines, screening paradigms, reagent types and
read-out methodologies, we explore in particular the complexities of
designing optimal controls and normalization strategies for these
challenging but extremely powerful studies.
RNA interference: PCR strategies for the quantification of stable degradation-fragments derived from siRNA-targeted mRNAs Peter Hahn, Cornelia Schmidt, Martin Weber, Jie Kang, Wolfgang Bielke Biomolecular Engineering 21 (2004) 113–117 mRNA targeted by siRNA
is endogeneously cleaved into a 5- and a 3-fragment and finally
degraded in cells. Little is known about the
relative stability and
degradation kinetics of these 5- and 3-fragments after the siRNA
mediated first cut. We present a qRT-PCR Protocol which allows the
determination of the optimal time point for mRNA analyses, helping to
avoid the generation of false positive effects in downstream
experiments, such as microarray analysis, which may be caused by
undegraded fragments of a siRNA-targeted mRNA.
Defining and Assaying RNAi in Mammalian Cells Konrad Huppi, Scott E. Martin, and Natasha J. Caplen* Gene Silencing Section Molecular Cell, Vol. 17, 1–10, January 7, 2005 The investigation of
protein function through the inhibition of activity has been critical
to our understanding of many normal and abnormal biological processes.
Until recently, functional inhibition in biological systems has been
induced using a variety of approaches including small molecule
antagonists, antibodies, aptamers, ribozymes, antisense
oligonucleotides or transcripts, morpholinos, dominant-negative
mutants, and knockout transgenic animals. Although all of these
approaches have made substantial advances in our understanding of the
function of many proteins, a lack of specificity or restricted
applicability has limited their utility. Recently, exploitation of the
naturally occurring posttranscriptional gene silencing mechanism
triggered by double-stranded RNA (dsRNA), termed RNA interference
(RNAi), has gained much favor as an alter-native means for analyzing
gene function. Aspects of the basic biology of RNAi, its application as
a functional genomics tool, and its potential as a therapeutic approach
have been extensively reviewed (Hannon and Rossi, 2004; Meister and
Tuschl, 2004); however, there has been only limited discussion as to
how to design and validate an individual RNAi effector molecule and how
to interpret RNAi data overall, particularly with reference to
experimentation in mammalian cells. This perspective will aim to
consider some of the issues encountered when conducting and
interpreting RNAi experiments in mammalian cells.
Designing siRNA That Distinguish between Genes That Differ by a Single Nucleotide Dianne S. Schwarz, Hongliu Ding, Lori Kennington, Jessica T. Moore, Janell Schelter, Julja Burchard, Peter S. Linsley, Neil Aronin, Zuoshang Xu, Phillip D. Zamore 1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 2 Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 3 Rosetta Inpharmatics, Merck and Co., Seattle, Washington, United States of America PLoS Genetics (2006) Volume 2 Issue 9 e140 1307 Small interfering RNAs
(siRNAs), the guides that direct RNA interference (RNAi), provide a
powerful tool to reduce the expression of a single gene in human cells.
Ideally, dominant, gain-of-function human diseases could be treated
using siRNAs that specifically silence the mutant disease allele, while
leaving expression of the wild-type allele unperturbed. Previous
reports suggest that siRNAs can be designed with single nucleotide
specificity, but no rational basis for the design of siRNAs with single
nucleotide discrimination has been proposed. We systematically
identified siRNAs that discriminate between the wild-type and mutant
alleles of two disease genes: the human Cu, Zn superoxide dismutase
(SOD1) gene, which contributes to the progression of hereditary
amyotrophic lateral sclerosis through the gain of a
toxic property, and the
huntingtin (HTT) gene, which causes Huntington disease when its
CAG-repeat region expands beyond approximately 35 repeats. Using
cell-free RNAi reactions in Drosophila embryo lysate and reporter
assays and microarray analysis of off-target effects in cultured human
cells, we identified positions within an siRNA that are most sensitive
to mismatches. We also show that purine:purine mismatches imbue an
siRNA with greater discriminatory power than other types of base
mismatches. siRNAs in which either a G:U wobble or a mismatch is
located in the ‘‘seed’’ sequence, the specialized siRNA guide region
responsible for target binding, displayed lower levels of selectivity
than those in which the mismatch was located 39 to the seed; this
region of an siRNA is critical for target cleavage but not siRNA
binding. Our data suggest that siRNAs can be designed to discriminate
between the wild-type and mutant alleles of many genes that differ by
just a single nucleotide.
|