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AG 105 Tuschl: siRNA 1999), we have systematically analyzed the silencing efficiency of siRNA duplexes as a function of the length of the siRNAs, the length of the overhang and the sequence in the overhang (Elbashir et al. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs. The antisense siRNA is synthesized as the complement to position 1 to 21 of the 23-nt motif. www.rockefeller.edu /labheads/tuschl/sirna.html   (3337 words)

Small interfering RNA - Wikipedia, the free encyclopedia Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long RNA molecules that play a variety of roles in biology. SiRNAs were first discovered by David Baulcombe's group in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants[1]. SiRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. en.wikipedia.org /wiki/SiRNA   (1319 words)

Ambion TechNotes 10(3): Silencer Validated siRNAs The cut-off for siRNA validation is a >70% reduction in target gene expression 48 hours post-transfection, though the average reduction in target gene expression in the inital set of validated siRNAs is greater than 90%. After annealing, the siRNAs are analyzed by nondenaturing gel or capillary electrophoresis to confirm that the strands are properly annealed. The indicated siRNAs were mixed with siPORT Lipid (Ambion) and the resulting complexes were added to HeLa cells in 24 well plates at the final concentration of siRNA shown. www.ambion.com /techlib/tn/103/3.html   (1435 words)

New HBV Therapy Research for siRNAs The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Two siRNA molecules, HBV263 and HBV1583, were selected and chemically stabilized for nuclease resistance using a previously reported method3 in which all 2'-OH residues on the RNA were substituted with 2'F, 2'O-Me or 2'H residues. Quantification of siRNA in plasma and liver samples was done using a sandwich hybridization assay with a working concentration range of 0.027-7.013 ng/ml for the sense strand and 0.026-6.663 ng/ml for the antisense strand. www.natap.org /2005/HBV/093005_02.htm   (3539 words)

Biocompare - Product Literature Center Following the siRNAs interaction with its substrate, the mRNA is targeted for degradation, perhaps by enzymes that are present in the RISC complex (Montgomery et al., 1998). The reduction in cell proliferation observed with the siRNA to the 3' UTR was similar to that found using the optimized antisense phosphorothioate oligonucleotide complementary to the start codon of the c-myc mRNA (Kimura et al., 1995). In contrast, siRNAs against the 5' UTR and Exon 2 of the c-myc mRNA affected cell proliferation similarly to the scrambled siRNA sequence, which was used as a negative control (Figure 2A). www.biocompare.com /techart.asp?id=323   (2162 words)

Sirna Therapeutics, Inc. :: Sirna Therapeutics Granted Patents Broadly Covering siRNAs Targeting VEGF and HCV Sirna Therapeutics is a clinical-stage biotechnology company developing RNAi-based therapies for serious diseases and conditions, including age- related macular degeneration (AMD), hepatitis B and C, dermatology, asthma, Huntington's Disease, diabetes and oncology. For example, Sirna's ability to develop products and operate as a going concern is contingent upon having readily available cash to fund its operating programs and is subject to the escalating expenses and risks associated with the initiation of clinical trials and their potential outcomes. Sirna undertakes no obligation to revise or update any forward-looking statements in order to reflect events or circumstances that may arise after the date of this release. sev.prnewswire.com /biotechnology/20050711/NYM10211072005-1.html   (1123 words)

siRNA Target Designer We recommend that the effects of each siRNA be compared to those of a mock siRNA that has the same sequence composition as the active siRNA but having a sequence which is scrambled relative to the siRNA sequence (4). All siRNAs are sorted by the free energy of the 5' antisense region. Several recent papers(9, 11, 14) have shown that siRNA efficacy is correlated with the free energy of the 5' antisense region. www.promega.com /siRNADesigner/program   (1682 words)

Plant Methods | Full text | A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts siRNAs are short (~21nt), double stranded regions of RNA that are incorporated into a silencing complex within a plant cell and direct the sequence specific cleavage of homologous mRNAs. To determine the optimum concentration of siRNAs needed to silence luciferase expression in rice seedling protoplasts, a range of concentrations (0, 0.3, 1, 2, 3, 5, 6 and 10 μg) of siLUC were co-transformed into protoplasts with 5 μg of pLUC. The use of siRNAs and transient assays hold great promise for increasing the speed at which genes can be studied, bridging the gap between the large data sets coming from hi-throughput assays and the time consuming and laborious but absolutely essential outcomes of in planta investigations. www.plantmethods.com /content/2/1/13   (4280 words)

RNAi-Based Analysis of Gene Function | Cells were treated with each of the siRNAs long enough to substantially reduce the MAPK Mrna and protein levels. Their data suggested yes: limited complementarity to the siRNA strands appeared sufficient for a Mrna to be a target for that siRNA. For example, expression profiling has shown that the siRNAs that are the most potent silencers of the intended targets have a low GC content at the 5' end and high GC content at the 3' end of the antisense strand. www.nyas.org /ebriefreps/main.asp?intSubsectionID=255   (999 words)

In Vitro Synthesized Small Interfering RNAs Elicit RNA Interference in African Trypanosomes: AN IN VITRO AND IN VIVO ... The gray bar in the lower panel specifies the section of the gel that is shown in the upper panel with a reduced contrast. A, siRNAs were generated by annealing of base complementary RNAs either synthesized by in vitro transcription (T7) or synthesized by solid phase chemical synthesis. Thermodynamic stabilities were calculated as in Ref. 44 based on pentameric sequence intervals within the double-stranded parts of the siRNAs. www.jbc.org /cgi/content/full/280/21/20573   (4739 words)

A Small Interfering RNA Targeting Coxsackievirus B3 Protects Permissive HeLa Cells from Viral Challenge -- Ahn et al. ... Cells were pretreated with or without various siRNAs, including an unmatched control siRNA, prior to infection with CVB3w-GFP under the conditions described in the legend to Fig. Following treatment with POL siRNA, HeLa cells were challenged with a variety of enteroviruses and examined for cytotoxicity through methyl thiazolyl tetrazolium assay. The POL siRNA sequence was compared with the corresponding sequences of other viruses. jvi.asm.org /cgi/content/full/79/13/8620   (1893 words)

Tips For Designing Your Own siRNAs - Biocompare Buying Tips The initial work on RNA interference from the labs of Tom Tuschl and others suggests that silencing is most effective when using double-stranded siRNA that is comprised of a 19-nt region that is complementary to the target gene, plus a 2-nt overhang at each 3’ end. It is generally agreed that siRNAs with 30-50% GC content are more effective than those with a higher or lower GC content, although the Tuschl lab finds that a GC content of up to 70% can be effective as well. One common type of control is a negative control consisting of your siRNA with a scrambled nucleotide sequence (with the nucleotide composition retained). biocompare.com /tips.asp?id=22   (1831 words)

Dicing Up RNAs. The antisense strand of the siRNA (blue) forms a continuous hybrid with the mRNA target (red), which is then degraded. Genetic and molecular analysis of proteins required for RNAi reveals that the core RNAi pathway (including members of the RDE family of RNA-binding proteins) evolved before the divergence of plants and animals (3). Proof that Dicer activity is required for RNAi comes from studies where loss of Dicer--by depleting Drosophila cells of the protein (1) or inactivating the dcr-1 gene in worms (2, 14)--results in the disabling of RNAi. www.euchromatin.org /Ambros01.htm   (1638 words)

In vivo activity of nuclease-resistant siRNAs -- LAYZER et al. 10 (5): 766 -- RNA siRNA quantities at various time points were calculated by dividing the total counts of full-length siRNA by the input starting material. The stability of 2'-F (squares) and 2'-OH (triangles) siRNAs in human plasma are compared over a period of 48 h. siRNA duplex were used with a final volume of 360 µL. Each experiment was repeated in triplicate. www.rnajournal.org /cgi/content/full/10/5/766   (3246 words)

Illuminating the Structure & Function of siRNA Researchers studying small inhibitory RNAs (siRNAs), which are used in a cellular process known as RNA interference (RNAi), want to learn which structural elements of siRNAs influence their function in human cells. By modifying the siRNAs, then watching how such changes affected cell fluorescence, the researchers assessed which aspects of siRNA structure contributed to its function, known as silencing. The studies showed that the cells attached phosphoryl groups to one end of the siRNAs; preventing this phosphorylation from occurring on the "antisense" strand (the strand complementary to the "sense," or coding, strand that is translated to form proteins) blocked the siRNAs' silencing function. www.nyas.org /ebrief/miniEB.asp?eBriefID=383   (269 words)

Small Interfering RNAs: A Revolutionary Tool for the Analysis of Gene Function and Gene Therapy -- Tuschl and Borkhardt ... The siRNA duplexes are incorporated into a siRNA-containing ribonucleoprotein complex (siRNP) (21) becoming the RNA-induced silencing complex (RISC) endonuclease, which targets homologous mRNAs for degradation. Target regions are selected such that siRNA sequences may contain uridine residues in the 2-nt overhangs. The transcription is terminated by the encounter of a polythymidine tract (T5) after the incorporation of two to three uridine residues encoded by the T5 element. molinterv.aspetjournals.org /cgi/content/full/2/3/158   (4902 words)

Specific Inhibition of AQP1 Water Channels in Isolated Rat Intrahepatic Bile Duct Units by Small Interfering RNAs -- ... IBDUs Isolation and Transfection with siRNAs-- IBDUs, which are portions of intrahepatic bile ducts ranging in luminal diameter from 100 to 125 µm and in length from 0.6 transfected with siRNAs in the absence of the lipid carrier. The advantages of siRNA technology for effective and specific suppression of selected genes are becoming increasingly apparent. www.jbc.org /cgi/content/full/278/8/6268   (4159 words)

Small Interfering RNA-Mediated Gene Silencing in T Lymphocytes -- McManus et al. 169 (10): 5754 -- The Journal of ... In this assay, cationic lipid cotransfection of the mouse The lower left density plot depicts a typical expression profile of an ineffective siRNA, which is identical to nonspecific siRNA control (not shown). siRNAs were evaluated by titrating increasing amounts of the siRNAs during cationic lipid cotransfection. www.jimmunol.org /cgi/content/full/169/10/5754   (4995 words)

Antiviral Effects of Human Immunodeficiency Virus Type 1-Specific Small Interfering RNAs against Targets Conserved in ... Twenty-one-base-pair siRNA duplexes were selected from within a region of the HIV-1 NL4-3 transcripts for the gp41 gene, tat, rev, and nef. Supernatants containing progeny virions generated from HeLaCD4 cells transfected with HIV-1-specific siRNAs at 2 dpi were tested to determine the infectivity of the viral particles in a MAGI assay. HIV-1-specific siRNA data are for 293T cells cotransfected with HIV-1-specific siRNAs (multiplexed) and plasmid DNA for HIV-1 (in triplicate; the means and standard deviations are indicated). jvi.asm.org /cgi/content/full/78/24/13687   (5950 words)

Pre-designed siRNAs - BioForum Pre-designed could mean that siRNAs are designed before you order it (not verified), or siRNAs are experimentally verified. It is not a good idea to target a mutation hot spot unless you know the type of mutation in a certain cell, because any mismatch between the siRNA and its target potentially affects its function depending on where the mismatch occurs. I just wanted to warn you about the G3PDH positive control that Ambion uses with their pSilencer kit -- it doesn't work! I went round and round with them, was repeatedly told that it was a transfection problem, but they eventually said that it just doesn't work in rat and mouse cells. www.protocol-online.org /forums/index.php?showtopic=7541   (479 words)

Filtering of Ineffective siRNAs and Improved siRNA Design Tool -- Yiu et al. 21 (2): 144 -- Bioinformatics effective siRNAs for human genes that are published in the literature. siRNAs from other candidates is generated with the help of AI techniques, in particular, the decision tree and support vector of centrosome amplification in cells depleted of Plk1 by siRNA. bioinformatics.oxfordjournals.org /cgi/content/full/21/2/144   (3810 words)

Highly specific and accurate selection of siRNAs for high-throughput functional assays -- Santoyo et al. 21 (8): 1376 ... Candidate siRNAs obtained using the default rules, except the score (Reynolds et al., 2004) which is set to 5. (2004); 62 siRNAs and 6 genes by Ui-Tei et al. (2000) and 46 siRNAs by Amarzguioui and Prydz (2004)] to identify bioinformatics.oxfordjournals.org /cgi/content/full/21/8/1376   (3886 words)

Control of translation and mRNA degradation by miRNAs and siRNAs -- Valencia-Sanchez et al. 20 (5): 515 -- Genes and ... Allen, E., Xie, Z., Gustafson, A.M., and Carrington, J.C. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., and Tuschl, T. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. www.genesdev.org /cgi/content/full/20/5/515   (6244 words)

Lake Tahoe 2005 siRNAs and miRNAs Fluorescent microscopy will be utilized to examine the efficiency of tranfection of cultured mammalian cells with custom designed siRNAs using immunofluorescence to evaluate the extent of knockdown of targeted gene expression. To highlight other important experimental procedures encountered in the use of siRNA to knock down gene expression, video presentations have been prepared and will be shown during the program. To further contribute to the success of your particular research program utilizing RNA interference, we have scheduled two round-table discussions. www.biotrac.com /pages/TahoesiRNAsandmiRNAs.html   (333 words)

siRNAs: a new wave of RNA-based therapeutics -- Coburn and Cullen 51 (4): 753 -- Journal of Antimicrobial Chemotherapy siRNAs: a new wave of RNA-based therapeutics -- Coburn and Cullen 51 (4): 753 -- Journal of Antimicrobial Chemotherapy vectors that are capable of expressing siRNAs in a stable manner The appearance of a resistant strain of poliovirus in cultures jac.oxfordjournals.org /cgi/content/full/51/4/753   (1975 words)

Cancer Therapy - Targeted delivery of siRNAs mediated by aptamer-siRNA chimeras "Technologies that mediate targeted delivery of siRNAs are needed to improve their therapeutic efficacy and safety. J.O. Mcnamara and colleagues, Duke University, explained, "The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. Higher levels of cells originating in bone marrow may be useful in prognosis of liver cancer www.newsrx.com /health-articles/366866.html   (460 words)

CiteULike: Knocking down Disease with siRNAs   (Site not responding. Last check: 2007-11-02) Knocking down expression of disease-related genes using small interfering RNAs (siRNAs) has potential for treating a variety of illnesses. This Essay will examine the opportunities for harnessing RNA interference (RNAi) for therapy, as well as the obstacles and possible ways to circumvent them. @article{citeulike:779141, abstract = {Knocking down expression of disease-related genes using small interfering RNAs (siRNAs) has potential for treating a variety of illnesses. www.citeulike.org /user/dchughes/article/779141   (110 words)

Gene Silencing by SiRNAs and Ribozymes   (Site not responding. Last check: 2007-11-02) Use your back button to return to previous Large scale experimental testing both in vitro and in vivo for validation of RNA target accessibility prediction (Ding and Lawrence 2001) for the design of short interfering RNAs (siRNAs) and trans-cleaving ribozymes. The results will be used to further develop methods for the rational design of RNA-targeting nucleic acids and will be incorporated into a RNA folding software named Sfold. www.wadsworth.org /resnres/bios/genesil.htm   (158 words)

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