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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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Monitoring JNK pathway activation. The puc gene is a direct target of the JNK pathway and is commonly used as a reporter for JNK activation (Boutros et al. 2002). A transient response of puckered (down after 2-hr stimulation) upon LPS stimulation in a Drosophila S2 cell line was observed. Wnt5 serves as a negative control. Two independent puckered qPCR primer pairs (PP19428 and PP31584) in FlyPrimerBank show a similar trend in response.
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fig5: Monitoring JNK pathway activation. The puc gene is a direct target of the JNK pathway and is commonly used as a reporter for JNK activation (Boutros et al. 2002). A transient response of puckered (down after 2-hr stimulation) upon LPS stimulation in a Drosophila S2 cell line was observed. Wnt5 serves as a negative control. Two independent puckered qPCR primer pairs (PP19428 and PP31584) in FlyPrimerBank show a similar trend in response.

Mentions: We used primers in FlyPrimerBank to monitor activation of the JNK signaling pathway (Figure 5). Activation of the JNK pathway has been extensively studied in Drosophila cell culture (Bond et al. 2008; Park et al. 2004; Pereira et al. 2011; Stronach 2005). Various endogenous or immune stimuli can activate the pathway, including LPS, components of the cell wall of gram-negative bacteria. The puckered (puc) gene is a direct target of the JNK pathway and transcriptional up-regulation of puc is commonly used as a reporter of JNK activation. We assessed JNK pathway stimulation in Drosophila S2 cells by monitoring puc expression following the addition of LPS to culture media for 30 min, 1 hr, and 2 hr. Wnt5, a ligand of the Wnt/Wg signaling pathway, whose regulation is considered independent of JNK pathway activity in this cell line and expression level is comparable with puc, was used as a negative control. The transcriptional levels of puc and Wnt5 were analyzed using SYBR Green-based qPCR analysis with primer pairs from FlyPrimerBank, validated using the same primer analysis procedure and acceptance criteria described previously. We observed similar levels of transient up-regulation of puc expression (down after a 2-hr stimulation) with two independent primer pairs. Meanwhile the transcript level of Wnt5 remained stable during the time course. The rapid up-regulation of puc expression detected using our qPCR assay is characteristic of the LPS transcriptional response in the S2 Drosophila cell line (Park et al. 2004).


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)

Monitoring JNK pathway activation. The puc gene is a direct target of the JNK pathway and is commonly used as a reporter for JNK activation (Boutros et al. 2002). A transient response of puckered (down after 2-hr stimulation) upon LPS stimulation in a Drosophila S2 cell line was observed. Wnt5 serves as a negative control. Two independent puckered qPCR primer pairs (PP19428 and PP31584) in FlyPrimerBank show a similar trend in response.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Monitoring JNK pathway activation. The puc gene is a direct target of the JNK pathway and is commonly used as a reporter for JNK activation (Boutros et al. 2002). A transient response of puckered (down after 2-hr stimulation) upon LPS stimulation in a Drosophila S2 cell line was observed. Wnt5 serves as a negative control. Two independent puckered qPCR primer pairs (PP19428 and PP31584) in FlyPrimerBank show a similar trend in response.
Mentions: We used primers in FlyPrimerBank to monitor activation of the JNK signaling pathway (Figure 5). Activation of the JNK pathway has been extensively studied in Drosophila cell culture (Bond et al. 2008; Park et al. 2004; Pereira et al. 2011; Stronach 2005). Various endogenous or immune stimuli can activate the pathway, including LPS, components of the cell wall of gram-negative bacteria. The puckered (puc) gene is a direct target of the JNK pathway and transcriptional up-regulation of puc is commonly used as a reporter of JNK activation. We assessed JNK pathway stimulation in Drosophila S2 cells by monitoring puc expression following the addition of LPS to culture media for 30 min, 1 hr, and 2 hr. Wnt5, a ligand of the Wnt/Wg signaling pathway, whose regulation is considered independent of JNK pathway activity in this cell line and expression level is comparable with puc, was used as a negative control. The transcriptional levels of puc and Wnt5 were analyzed using SYBR Green-based qPCR analysis with primer pairs from FlyPrimerBank, validated using the same primer analysis procedure and acceptance criteria described previously. We observed similar levels of transient up-regulation of puc expression (down after a 2-hr stimulation) with two independent primer pairs. Meanwhile the transcript level of Wnt5 remained stable during the time course. The rapid up-regulation of puc expression detected using our qPCR assay is characteristic of the LPS transcriptional response in the S2 Drosophila cell line (Park et al. 2004).

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