FlyPrimerBank: An Online Database for <i>Drosophila melanogaster</i> Gene Expression Analysis and Knockdown Evaluation of RNAi Reagents

Hu, Yanhui; Sopko, Richelle; Foos, Marianna; Kelley, Colleen; Flockhart, Ian; Ammeux, Noemie; Wang, Xiaowei; Perkins, Lizabeth A.; Perrimon, Norbert; Mohr, Stephanie E.

doi:10.1534/g3.113.007021

Cited by 144 publications

(109 citation statements)

References 34 publications

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“…For the selected reference genes (α-Tub84b, eif-1a, and CG13220), we next confirmed that the presence plot for each gene matched its corresponding FlyBase model (St Pierre et al 2014). We then selected primers using FlyPrimerBank (Hu et al 2013) and relevant publications (Ling and Salvaterra 2011) and optimized them for qRT-PCR.…”

Section: Reference Genesmentioning

confidence: 63%

“…For primer optimization (Eurofins MWG Operon), we produced standard curves using at least five 1:5 dilutions of RNA starting at 50 ng cDNA. Primer sequences were taken from FlyPrimerBank and citations (Ling and Salvaterra 2011;Hu et al 2013). Gene names, primer sequences, amplicon length, efficiency, R 2 of primers are given in Supplemental Table S9.…”

Section: Qrt-pcrmentioning

confidence: 99%

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Genomic responses to the socio-sexual environment in male Drosophila melanogaster exposed to conspecific rivals

Mohorianu

Bretman

Smith

et al. 2017

RNA

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Socio-sexual environments have profound effects on fitness. Local sex ratios can alter the threat of sexual competition, to which males respond via plasticity in reproductive behaviors and ejaculate composition. In Drosophila melanogaster, males detect the presence of conspecific, same-sex mating rivals prior to mating using multiple, redundant sensory cues. Males that respond to rivals gain significant fitness benefits by altering mating duration and ejaculate composition. Here we investigated the underlying genome-wide changes involved. We used RNA-seq to analyze male transcriptomic responses 2, 26, and 50 h after exposure to rivals, a time period that was previously identified as encompassing the major facets of male responses to rivals. The results showed a strong early activation of multiple sensory genes in the head-thorax (HT), prior to the expression of any phenotypic differences. This gene expression response was reduced by 26 h, at the time of maximum phenotypic change, and shut off by 50 h. In the abdomen (A), fewer genes changed in expression and gene expression responses appeared to increase over time. The results also suggested that different sets of functionally equivalent genes might be activated in different replicates. This could represent a mechanism by which robustness is conferred upon highly plastic traits. Overall, our study reveals that mRNA-seq can identify subtle genomic signatures characteristic of flexible behavioral phenotypes.

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Section: Reference Genesmentioning

confidence: 63%

Section: Qrt-pcrmentioning

confidence: 99%

Genomic responses to the socio-sexual environment in male Drosophila melanogaster exposed to conspecific rivals

Mohorianu

Bretman

Smith

et al. 2017

RNA

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“…RT-qPCR was performed using QScript XLT 1-step RT-qPCR ToughMix (Quanta Biosciences) on a CFX96 Touch RealTime PCR Detection system (Bio-Rad) with a commercially designed primer and probe assay (TaqMan Gene Expression Assays, Life Technologies). Alternatively, first-strand cDNA was synthesized with random nonamer primers before qPCR using iQ SYBR Green Supermix (Bio-Rad), as described previously (66), and predefined primer pairs from FlyPrimerBank (67). Relative gene expression was calculated using the ΔΔCt method (68) and normalized to total input RNA.…”

Section: Methodsmentioning

confidence: 99%

The 4E-BP growth pathway regulates the effect of ambient temperature on Drosophila metabolism and lifespan

Carvalho

Drago

Hoxha

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Changes in body temperature can profoundly affect survival. The dramatic longevity-enhancing effect of cold has long been known in organisms ranging from invertebrates to mammals, yet the underlying mechanisms have only recently begun to be uncovered. In the nematode Caenorhabditis elegans, this process is regulated by a thermosensitive membrane TRP channel and the DAF-16/FOXO transcription factor, but in more complex organisms the underpinnings of cold-induced longevity remain largely mysterious. We report that, in Drosophila melanogaster, variation in ambient temperature triggers metabolic changes in protein translation, mitochondrial protein synthesis, and posttranslational regulation of the translation repressor, 4E-BP (eukaryotic translation initiation factor 4E-binding protein). We show that 4E-BP determines Drosophila lifespan in the context of temperature changes, revealing a genetic mechanism for cold-induced longevity in this model organism. Our results suggest that the 4E-BP pathway, chiefly thought of as a nutrient sensor, may represent a master metabolic switch responding to diverse environmental factors.S tudies on the biological underpinnings of aging have uncovered numerous genetic and environmental factors regulating animal lifespan. Longevity-promoting genes include components of the insulin-like signaling pathway, the histone deacetylase Sir2, the GTPase Ras, TRP membrane channels, and transcription factors, among many others (1, 2). Environmental manipulations extending life include changes in nutrition (often referred to as caloric or dietary restriction), sexual/reproductive history, and ambient temperature (3-5).Of the factors known to impact aging, temperature is arguably the most promising. Lifespan extension by cold is evolutionarily conserved-documented in poikilotherms, such as worms and flies, and homeotherms, including mammals (4, 6-11)-and more robust than well-studied interventions such as dietary restriction (12, 13). However, the underlying mechanisms remain incompletely understood (10,14,15). In Caenorhabditis elegans, the effect of cold on survival involves the thermosensitive membrane channel TRPA-1 acting upstream of the DAF-16/FOXO transcription factor (14,15). This seminal finding countered the notion that longevity is the passive result of general thermodynamic changes, and favored instead the view that genetic pathways actively control lifespan in response to ambient temperature. However, these results have to date not been extended or replicated in other model organisms. Explanatory theories for the effect of cold on longevity include a reduction in reactive oxygen species generated by mitochondrial uncoupling proteins, suppression of autoimmune response, and changes in neuroendocrine factors (6,16,17), but these views remain largely speculative given the lack of further mechanistic insight into lifespan extension by cold in model organisms.We report that, in Drosophila, changes in temperature trigger a metabolic program impacting protein translation, mitochondrial prot...

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“…On average, 65% of TRiP stocks display knockdown efficiencies of .50% ( Figure 6A). To facilitate searching for primers appropriate for RT-qPCR analysis, we assembled FlyPrimerBank (http://www.flyrnai.org/ FlyPrimerBank) (Hu et al 2013b). Relevant to long dsRNA reagents, the tool indicates if a given primer pair should be avoided as the primers are predicted to amplify the dsRNA itself.…”

Section: Validation Of the Trip Linesmentioning

confidence: 99%

The Transgenic RNAi Project at Harvard Medical School: Resources and Validation

et al. 2015

Self Cite

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To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China). KEYWORDS RNAi; Drosophila; screens; phenotypes; functional genomics A striking finding from the genomic revolution and wholegenome sequencing is the amount of information missing on gene function. Although Drosophila is arguably the bestunderstood multicellular organism and a proven model system for human diseases, mutations mapped to specific genes with readily detectable phenotypes have been isolated for 15% of the .13919 annotated fly coding genes (http:// flybase.org/; FlyBase R6.06). The lack of information on the majority of genes (the "phenotype gap") suggests that researchers have been unable to either assay their roles experimentally and/or resolve issues of functional redundancy. In addition, some phenotypes may be only detected on specific diets and environments. Further, our understanding of the function of many genes for which we have some information is limited by pleiotropy, whereby an earlier function of the gene prevents analysis of later functions.The availability of in vivo RNAi has revolutionized the ability of Drosophila researchers to disrupt the activity of single genes with spatial and temporal resolution (Dietzl et al. 2007; see review by Perrimon et al. 2010), and thus address the phenotype gap. Motivated by the power of the approach and the needs of the community, three large-scale efforts, the Vienna Drosophila RNAi Center (VDRC, http:// stockcenter.vdrc.at/control/main), the National Institute of Genetics (NIG, http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp), and the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) (http://www.flyrnai.org/TRiP-HOME. html) have over the years generated large numbers of RNAi lines that aim to cover all Drosophila genes. These resources are proving invaluable to address a myriad of questions in various biological and biomedical fields including but not limite...

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FlyPrimerBank: An Online Database for Drosophila melanogaster Gene Expression Analysis and Knockdown Evaluation of RNAi Reagents

Cited by 144 publications

References 34 publications

Genomic responses to the socio-sexual environment in male Drosophila melanogaster exposed to conspecific rivals

Genomic responses to the socio-sexual environment in male Drosophila melanogaster exposed to conspecific rivals

The 4E-BP growth pathway regulates the effect of ambient temperature on Drosophila metabolism and lifespan

The Transgenic RNAi Project at Harvard Medical School: Resources and Validation

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