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FlyPrimerBank: an online database for Drosophila melanogaster gene expression analysis and knockdown evaluation of RNAi reagents.

Hu Y, Sopko R, Foos M, Kelley C, Flockhart I, Ammeux N, Wang X, Perkins L, Perrimon N, Mohr SE - G3 (Bethesda) (2013)

Bottom Line: More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown.Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial.All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank).

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115.

ABSTRACT
The evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo.

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Observed RNAi knockdown levels are independent of primer design.
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fig4: Observed RNAi knockdown levels are independent of primer design.

Mentions: We next evaluated the utility of primers in FlyPrimerBank to assess knockdown in transgenic fly lines bearing shRNAs targeting embryonic Drosophila protein kinases and phosphatases (KPs). We first assembled a target list of KP genes. A few studies have been performed to identify Drosophila KPs on a genome-wide level using sequence comparison-based algorithms (Manning et al. 2002; Morrison et al. 2000). To supplement the gene list from these publications, we mined Drosophila structural and functional gene annotations from public databases. Additionally, we mapped human KP genes to fly genes using DIOPT, an ortholog prediction tool (Hu et al. 2011). The assembled KP list contains 268 kinase and 112 phosphatases (Table S2). Based on modEncode RNA-seq analysis (Graveley et al. 2011), we selected 474 TRiP fly stocks bearing shRNAs targeting 344 KPs that are expressed (FPKM > 3) in early Drosophila embryos for knockdown assessment. To measure transcripts in early embryos (0−4 hr) expressing a unique KP-targeting shRNA as compared to embryos expressing a control shRNA targeting EGFP, we established a medium-throughput pipeline again compliant with the MIQE guidelines (Bustin et al. 2009). For 27 lines, we assessed transcript levels using two different primer pairs. Linear regression of knockdown levels of this subset generated an R-squared value of 0.8 (Pearson correlation co-efficiency was 0.9; Figure 4), indicating that primers of independent designs led to similar conclusions regarding the level of knockdown. Furthermore, our analysis revealed that 60% of the transgenic lines tested achieved 60% or more down-regulation of their intended target transcript(s). Moreover, we found that in nearly all cases for which the shRNA was associated with an embryonic phenotype, we measured more than 60% knockdown of the intended transcript (Sopko, Foos, Binari, Perkins and Perrimon, unpublished data), further substantiating our primer design and qPCR testing approach. The lines that failed to achieve 60% knockdown are highly enriched for shRNAs targeting UTRs (P = 0.004), likely reflecting inaccuracies in UTR annotation (Hu et al. 2013).


FlyPrimerBank: an online database for Drosophila melanogaster gene expression analysis and knockdown evaluation of RNAi reagents.

Hu Y, Sopko R, Foos M, Kelley C, Flockhart I, Ammeux N, Wang X, Perkins L, Perrimon N, Mohr SE - G3 (Bethesda) (2013)

Observed RNAi knockdown levels are independent of primer design.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3755921&req=5

fig4: Observed RNAi knockdown levels are independent of primer design.
Mentions: We next evaluated the utility of primers in FlyPrimerBank to assess knockdown in transgenic fly lines bearing shRNAs targeting embryonic Drosophila protein kinases and phosphatases (KPs). We first assembled a target list of KP genes. A few studies have been performed to identify Drosophila KPs on a genome-wide level using sequence comparison-based algorithms (Manning et al. 2002; Morrison et al. 2000). To supplement the gene list from these publications, we mined Drosophila structural and functional gene annotations from public databases. Additionally, we mapped human KP genes to fly genes using DIOPT, an ortholog prediction tool (Hu et al. 2011). The assembled KP list contains 268 kinase and 112 phosphatases (Table S2). Based on modEncode RNA-seq analysis (Graveley et al. 2011), we selected 474 TRiP fly stocks bearing shRNAs targeting 344 KPs that are expressed (FPKM > 3) in early Drosophila embryos for knockdown assessment. To measure transcripts in early embryos (0−4 hr) expressing a unique KP-targeting shRNA as compared to embryos expressing a control shRNA targeting EGFP, we established a medium-throughput pipeline again compliant with the MIQE guidelines (Bustin et al. 2009). For 27 lines, we assessed transcript levels using two different primer pairs. Linear regression of knockdown levels of this subset generated an R-squared value of 0.8 (Pearson correlation co-efficiency was 0.9; Figure 4), indicating that primers of independent designs led to similar conclusions regarding the level of knockdown. Furthermore, our analysis revealed that 60% of the transgenic lines tested achieved 60% or more down-regulation of their intended target transcript(s). Moreover, we found that in nearly all cases for which the shRNA was associated with an embryonic phenotype, we measured more than 60% knockdown of the intended transcript (Sopko, Foos, Binari, Perkins and Perrimon, unpublished data), further substantiating our primer design and qPCR testing approach. The lines that failed to achieve 60% knockdown are highly enriched for shRNAs targeting UTRs (P = 0.004), likely reflecting inaccuracies in UTR annotation (Hu et al. 2013).

Bottom Line: More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown.Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial.All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank).

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115.

ABSTRACT
The evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo.

Show MeSH