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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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Primers that failed qPCR testing as a function of intron size.
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fig3: Primers that failed qPCR testing as a function of intron size.

Mentions: We next asked whether primer failure correlated with any features of the PCR amplified regions. We found enrichment among “failed” primers for primers that span exon:exon junctions where the spliced intron is small (Figure 3). In addition, with a bigger dataset that includes results from additional studies (R. Sopko, personal communication), we found that primers corresponding to genes expressed at low levels as well as genes that are “poorly” understood (i.e., genes associated with smaller numbers of Gene Ontology terms or publications) are more likely to fail than the primers targeting the genes expressed at high levels and/or relatively well-studied genes (i.e., genes with more Gene Ontology terms and publications).


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)

Primers that failed qPCR testing as a function of intron size.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Primers that failed qPCR testing as a function of intron size.
Mentions: We next asked whether primer failure correlated with any features of the PCR amplified regions. We found enrichment among “failed” primers for primers that span exon:exon junctions where the spliced intron is small (Figure 3). In addition, with a bigger dataset that includes results from additional studies (R. Sopko, personal communication), we found that primers corresponding to genes expressed at low levels as well as genes that are “poorly” understood (i.e., genes associated with smaller numbers of Gene Ontology terms or publications) are more likely to fail than the primers targeting the genes expressed at high levels and/or relatively well-studied genes (i.e., genes with more Gene Ontology terms and publications).

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