Naito, Y. et al. dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference. Nucleic Acids Res. 33, W589-W591
ABSTRACT 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 short-interfering RNA (siRNA) cocktails. The software simulates this process and investigates individual 19 nt substrings of the long dsRNA. 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 'off-target minimized' dsRNA design, which is essential for reliable experiments in RNAi-based functional genomics.
Full-textDOI: · Available from: Kumiko Ui-Tei, Sep 29, 2015
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- "To ensure dsRNA specificity and to exclude potential non-specific targets, we used the E-RNAi , dsCheck  and DEQOR  programs to determine the dsRNA sequence. However, because the A. fluviatilis gene sequences are not available, we performed the analyses based on equivalent regions of the GSK-3 orthologous genes and genome sequences of other species (A. "
ABSTRACT: Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W-) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism.PLoS ONE 06/2014; 9(6):e98966. DOI:10.1371/journal.pone.0098966 · 3.23 Impact Factor
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- "However, to reinforce this inactivation, we introduced either another UAS- zfh-2RNAi insertion or the UAS-Dicer-2 (Dcr-2) construct (Dietzl et al., 2007) into some of our mutant combinations. The two RNA interference constructs are directed against different regions of the zfh-2 RNA (data not shown) and the RNAi construct previously described (Terriente et al., 2008) was checked with the DsCheck software (Naito et al., 2005; http://dscheck.rnai.jp) for possible offtargets (with 0 mismatches) and found to have none. "
ABSTRACT: The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.Developmental Biology 10/2013; 385(2). DOI:10.1016/j.ydbio.2013.10.011 · 3.55 Impact Factor
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- "This compares the target sequences with genome sequences of Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Oryza sativa and Rattus Norvegicus from the database and identifies dissimilar regions (http://dsCheck.RNAi.jp/) (Naito et al., 2005). Further, the sequence of the selected region was analysed for minimizing the possible homology with other gene sequences of H. armigera using NCBI-BLAST. "
ABSTRACT: Ribonucleic acid interference (RNAi) is a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA). Recently, RNAi has gained popularity as a reverse genetics tool owing to its tremendous potential in insect pest management, which includes Helicoverpa armigera. However, its efficiency is mainly governed by dsRNA concentration, frequency of application, target gene, etc. Therefore, to obtain a robust RNAi response in H. armigera, we evaluated various concentrations of dsRNA and its frequency of applications delivered through diet in silencing a midgut gene, chymotrypsin and a non-midgut gene, juvenile hormone acid methyl transferase (jhamt) of H. armigera. The extent of target gene silencing was determined by employing reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Our study revealed four significant findings: (i) single application of dsRNA elicited a delayed and transient silencing, while multiple applications resulted in early and persistent silencing of the above genes; (ii) silencing of the non-midgut gene (jhamt) through diet delivered dsRNA revealed prevalence of systemic silencing probably due to communication of silencing signals in this pest; (iii) the extent of silencing of chymotrypsin was positively correlated with dsRNA concentration and was negatively correlated with jhamt; (iv) interestingly, over-expression (15-18 folds) of an upstream gene, farnesyl diphosphate synthase (fpps), in juvenile hormone (JH) biosynthetic pathway at higher concentrations of jhamt dsRNA was the plausible reason for lesser silencing of jhamt. This study provides an insight into RNAi response of target genes, which is essential for RNAi design and implementation as a pest management strategy.Bulletin of entomological research 04/2013; 103:555-563. DOI:10.1017/S0007485313000138. · 1.91 Impact Factor