RNA interference ( RNAi )   &   basic and essential siRNA publication (2)
siRNA   &   quantitative real-time RT-PCR (1)
siRNA   &   quantitative real-time RT-PCR (3)
siRNA   &   quantitative real-time RT-PCR (4)

Basic and essential RNAi  &  siRNA publications:

Basic and essential RNAi & siRNA publications:

A species of small antisense RNA in posttranscriptional gene silencing in plants.

Hamilton AJ, Baulcombe DC.
Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
Science. 1999 286(5441): 950-952.

Posttranscriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs. Here, three types of transgene-induced PTGS and one example of virus-induced PTGS were analyzed in plants. In each case, antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an RNA template and may represent the specificity determinant of PTGS.

RNA silencing in plants.
David Baulcombe
Nature 431(2004), p. 356-363
There are at least three RNA silencing pathways for silencing specific genes in plants. In these pathways, silencing signals can be amplified and transmitted between cells, and may even be self-regulated by feedback mechanisms. Diverse biological roles of these pathways have been established, including defence against viruses, regulation of gene expression and the condensation of chromatin into heterochromatin. We are now in a good position to investigate the full extent of this functional diversity in genetic and epigenetic mechanisms of genome control.

DNA events:   An RNA microcosm.
Baulcombe D.
Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK.

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
  Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T.
  Nature. 2001 411(6836):494-8.
  Department of Cellular Biochemistry, Max-Planck-Institute for Biophysical
  Chemistry, Gottingen, Germany.
RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

RNA interference is mediated by 21- and 22-nucleotide RNAs.
Elbashir SM, Lendeckel W, Tuschl T.
Genes Dev. 2001 15(2):188-200.
Department of Cellular Biochemistry, Max-Planck-Institute for Biophysical
Chemistry, Am Fassberg 11, D-37077 Gottingen, Germany.

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex.

Analysis of gene function in somatic mammalian cells using small interfering RNAs.
Elbashir SM, Harborth J, Weber K, Tuschl T.
Methods. 2002 26(2): 199-213.

Department of Cellular Biochemistry, Max-Planck-Institute for Biophysical
Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
RNA interference (RNAi) is a highly conserved gene silencing mechanism that uses double-stranded RNA (dsRNA) as a signal to trigger the degradation of homologous mRNA. The mediators of sequence-specific mRNA degradation are 21- to 23-nt smallinterfering RNAs (siRNAs) generated by ribonuclease III cleavage from longerdsRNAs. Twenty-one-nucleotide siRNA duplexes trigger specific gene silencing inmammalian somatic cells without activation of the unspecific interferonresponse. Here we provide a collection of protocols for siRNA-mediated knockdownof mammalian gene expression. Because of the robustness of the siRNA knockdowntechnology, genomewide analysis of human gene function in cultured cells has now become possible.

Identification and characterization of small RNAs involved in RNA silencing.
Aravin A, Tuschl  T.
FEBS Lett. 2005 579(26): 5830-5840.
Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA.

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.

siRNAs: applications in functional genomics and potential as therapeutics.
Dorsett Y, Tuschl T.
Nat Rev Drug Discov. 2004 3(4): 318-329.
Laboratory of RNA Molecular Biology, Rockefeller University, 1230 York Avenue,
Box 186, New York, New York 10021, USA.

Molecules that can specifically silence gene expression are powerful research tools. Much effort has been put into the development of such molecules and has resulted in the creation of different classes of potential therapeutic agents. Small interfering RNA (siRNA) is one of the latest additions to the repertoire of sequence-specific gene-silencing agents. The robustness of this approach has motivated numerous biotechnology organizations and academic institutions to develop siRNA libraries for high-throughput genome-wide screening in mammalian cells. This article first overviews current nucleic-acid-based approaches for gene silencing, and then focuses on the application of siRNAs in particular in functional genomics and as potential therapeutics.

On the art of identifying effective and specific siRNAs.
Pei Y, Tuschl T.
Nat Methods. 2006 3(9):670-6.
Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The
Rockefeller University, 1230 York Avenue, Box 186, New York, New York 10021, USA.

Small interfering RNAs (siRNAs) have been widely exploited for sequence-specific gene knockdown, predominantly to investigate gene function in cultured vertebrate cells, and also hold promise as therapeutic agents. Because not all siRNAs that are cognate to a given target mRNA are equally effective, computational tools have been developed based on experimental data to increase the likelihood of selecting effective siRNAs. Furthermore, because target-complementary siRNAs can also target other mRNAs containing sequence segments that are partially complementary to the siRNA, most computational tools include ways to reduce potential off-target effects in the siRNA selection process. Though these methods facilitate selection of functional siRNAs, they do not yet alleviate the need for experimental validation. This perspective provides a practical guide based on current wisdom for selecting siRNAs.

MINIREVIEW:   RNA interference and small interfering RNAs.
Tuschl T.
Chembiochem. 2001 2(4):239-45.

Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, 37070 Gottingen, Germany.

REVIEW:  Small interfering RNAs: a revolutionary tool for the analysis of gene function and gene therapy.
Tuschl T, Borkhardt A.
Mol Interv. 2002 2(3):158-167.
Department of Cellular Biochemistry, Max-Planck-Institute for Biophysical
Chemistry, D-37077 Goettingen, Germany.

RNA interference (RNAi) represents an evolutionarily conserved cellular defense for controlling the expression of foreign genes in most eukaryotes including humans. RNAi is triggered by double-stranded RNA (dsRNA) and causessequence-specific mRNA degradation of single-stranded targetRNAs homologous in response to dsRNA. The mediators ofmRNA degradation are small interfering RNA duplexes (siRNAs), which are produced from long dsRNA by enzymatic cleavage inthe cell. siRNAs are approximately twenty-one nucleotides inlength, and have a base-paired structure characterized by twonucleotide3'-overhangs. Chemically synthesized siRNAs havebecome powerful reagents for genome-wide analysis ofmammalian gene function in cultured somatic cells. Beyondtheir value for validation of gene function, siRNAs also hold great potential as gene-specific therapeutic agents.

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.
Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA.
Proc Natl Acad Sci U S A. 2001 98(17):9742-7. Epub 2001 Jul 31.
Medical Genetics Branch, National Human Genome Research Institute, National
Institutes of Health, Bethesda, MD 20892, USA.

Short interfering RNAs (siRNAs) are double-stranded RNAs of approximately 21-25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristicstructure, with 5'-phosphate/3'-hydroxyl ends and a 2-base 3' overhang on eachstrand of the duplex. In this study, we present data that synthetic siRNAs caninduce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs wassuperior to the inhibition of gene expression mediated by single-strandedantisense oligonucleotides. The siRNAs seem to avoid the well documentednonspecific effects triggered by longer double-stranded RNAs in mammalian cells.These observations may open a path toward the use of siRNAs as a reverse geneticand therapeutic tool in mammalian cells.

siRNAs can function as miRNAs.
Doench JG, Petersen CP, Sharp PA.
Genes Dev. 2003 17(4):438- 442.
Center for Cancer Research, Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA.

With the discovery of RNA interference (RNAi) and related phenomena, new regulatory roles attributed to RNA continue to emerge. Here we show, in mammalian tissue culture, that a short interfering RNA (siRNA) can repress expression of a target mRNA with partially complementary binding sites in its 3' UTR, much like the demonstrated function of endogenously encoded microRNAs (miRNAs). The mechanism for this repression is cooperative, distinct from the catalytic mechanism of mRNA cleavage by siRNAs. The use of siRNAs to study translational repression holds promise for dissecting the sequence andstructural determinants and general mechanism of gene repression by miRNAs.

Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs.
Raymond CK, Roberts BS, Garrett-Engele P, Lim LP, Johnson JM.
RNA. 2005 11(11):1737-44.
Rosetta Inpharmatics, Seattle, WA 98109, USA.

There has been a surge of interest in the biology of microRNAs and the
technology of RNA interference. We describe a simple, robust, inexpensive assay for quantitative analysis of microRNAs and short-interfering RNAs. The method relies on primer extension conversion of RNA to cDNA by reverse transcription followed by quantitative, real-time PCR. Technical parameters critical to the success of the assay are presented. Measurements of microRNA levels are sensitive, with most assays allowing measurements in the femtomolar range, which corresponds to tens of copies per cell or less. The assay has a high dynamic range and provides linear readout over differences in microRNA concentrations that span 6-7 orders of magnitude. The assay is capable of discriminating between related microRNA family members that differ by subtle sequence differences. We used the method for quantitative analysis of six microRNAs across 12 tissue samples. The data confirm striking variation in the patterns of expression of these noncoding regulatory RNAs.

RNA interference: the molecular immune system.
Bagasra O, Prilliman KR.
J Mol Histol. 2004 35(6):545-53.
South Carolina Center for Biotechnology, Claflin University, Orangeburg, SC, USA.

Introduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS). The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer. siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo- and endo-nucleases as well as other proteins. Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner. RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals. In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression. In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21- to 23-nt-long siRNAs. However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi. siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells. Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements. Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described. Recent data in C. elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed. However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined. Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells. RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins. RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla. We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements. The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed.

A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst.
Nishikura K.
Cell. 2001 107(4):415-8.
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.

One of the many intriguing features of gene silencing by RNA interference is the apparent catalytic nature of the phenomenon. New biochemical and genetic evidence now shows that an RNA-directed RNA polymerase chain reaction, primed by siRNA, amplifies the interference caused by a small amount of "trigger" dsRNA.

Role for a bidentate ribonuclease in the initiation step of RNA interference.
Bernstein E, Caudy AA, Hammond SM, Hannon GJ.
Nature. 2001 409(6818): 363-6.
Cold Spring Harbor Laboratory, New York 11724, USA.

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are
degraded to generate new siRNAs.
Lipardi C, Wei Q, Paterson BM.
Cell. 2001 107(3):297-307.

Laboratory of Biochemistry, National Cancer Institute, National Institutes of
Health, Bethesda, MD 20892, USA.

In posttranscriptional gene silencing (PTGS), "quelling," and RNA interference (RNAi), 21-25 nucleotide RNA fragments are produced from the initiating dsRNA.
These short interfering RNAs (siRNAs) mediate RNAi by an unknown mechanism. Here, we show that GFP and Pp-Luc siRNAs, isolated from a protein complex in Drosophila embryo extract, target mRNA degradation in vitro. Most importantly, these siRNAs, as well as a synthetic 21-nucleotide duplex GFP siRNA, serve as primers to transform the target mRNA into dsRNA. The nascent dsRNA is degraded to eliminate the incorporated target mRNA while generating new siRNAs in a cycle of dsRNA synthesis and degradation. Evidence is presented that mRNA-dependent siRNA incorporation to form dsRNA is carried out by an RNA-dependent RNA polymerase activity (RdRP).

siRNA and miRNA: an insight into RISCs.
Tang G.
Trends Biochem Sci. 2005 30(2): 106-114.
Department of Biochemistry and Molecular Pharmacology, University of
Massachusetts Medical School, Worcester, MA 01605, USA.

Two classes of short RNA molecule, small interfering RNA (siRNA) and microRNA (miRNA), have been identified as sequence-specific posttranscriptional regulators of gene expression. siRNA and miRNA are incorporated into related RNA-induced silencing complexes (RISCs), termed siRISC and miRISC, respectively. The current model argues that siRISC and miRISC are functionally interchangeable and target specific mRNAs for cleavage or translational repression, depending on the extent of sequence complementarity between the small RNA and its target. Emerging evidence indicates, however, that siRISC and miRISC are distinct complexes that regulate mRNA stability and translation. The assembly of RISCs can be traced from the biogenesis of the small RNA molecules and the recruitment of these RNAs by the RISC loading complex (RLC) to the transition of the RLC into the active RISC. Target recognition by the RISC can then take place through different interacting modes.

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