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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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Primer testing pipeline and qPCR testing criteria.
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fig2: Primer testing pipeline and qPCR testing criteria.

Mentions: We established a primer-testing pipeline using cDNA isolated from early Drosophila embryos in compliance with the “Minimum Information for the publication of real-time Quantitative PCR Experiments” (i.e., MIQE) guidelines (bustin et al. 2009). Primers were tested by thermal analysis using SYBR Green-based qPCR as well as by size analysis using gel electrophoresis and sequencing after conventional PCR. To generate the cDNA template, embryos (0−4 hr) were collected and RNA was extracted, enriching for RNAs larger than 200 bps. Purified RNA was treated with DNAse and subsequently used for in vitro transcription to generate a cDNA library. cDNA was then serially diluted four times, starting with 1 μg of cDNA and decreasing the concentration with each dilution by a factor of four. R-squared values and primer efficiencies were calculated using Bio-Rad CFX Manager based on the results of a two-step qPCR program. Using preliminary data, we established acceptance criteria for assay performance (Figure 2), which included 90–120% PCR amplification efficiency, linear regression with R-squared values >0.995, and the following three visual features of amplification/melting calibration curves: (1) dilution curves are evenly distributed with two cycles separating each, indicative of a linear dynamic range; (2) the curve corresponding to the most diluted sample crosses a single threshold before cycle 30 and is at least five cycles away from a no template control (reaction mix and primers with no cDNA template); and (3) a single unique and sharp melting peak is observed (Supporting Information, Figure S1).


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)

Primer testing pipeline and qPCR testing criteria.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Primer testing pipeline and qPCR testing criteria.
Mentions: We established a primer-testing pipeline using cDNA isolated from early Drosophila embryos in compliance with the “Minimum Information for the publication of real-time Quantitative PCR Experiments” (i.e., MIQE) guidelines (bustin et al. 2009). Primers were tested by thermal analysis using SYBR Green-based qPCR as well as by size analysis using gel electrophoresis and sequencing after conventional PCR. To generate the cDNA template, embryos (0−4 hr) were collected and RNA was extracted, enriching for RNAs larger than 200 bps. Purified RNA was treated with DNAse and subsequently used for in vitro transcription to generate a cDNA library. cDNA was then serially diluted four times, starting with 1 μg of cDNA and decreasing the concentration with each dilution by a factor of four. R-squared values and primer efficiencies were calculated using Bio-Rad CFX Manager based on the results of a two-step qPCR program. Using preliminary data, we established acceptance criteria for assay performance (Figure 2), which included 90–120% PCR amplification efficiency, linear regression with R-squared values >0.995, and the following three visual features of amplification/melting calibration curves: (1) dilution curves are evenly distributed with two cycles separating each, indicative of a linear dynamic range; (2) the curve corresponding to the most diluted sample crosses a single threshold before cycle 30 and is at least five cycles away from a no template control (reaction mix and primers with no cDNA template); and (3) a single unique and sharp melting peak is observed (Supporting Information, Figure S1).

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