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.