title: FlyPrimerBank: an online database for Drosophila melanogaster gene expression analysis and knockdown evaluation of RNAi reagents.
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.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Hu
authors.forename: Richelle
authors.initials: R
authors.lastname: Sopko
authors.forename: Marianna
authors.initials: M
authors.lastname: Foos
authors.forename: Colleen
authors.initials: C
authors.lastname: Kelley
authors.forename: Ian
authors.initials: I
authors.lastname: Flockhart
authors.forename: Noemie
authors.initials: N
authors.lastname: Ammeux
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Wang
authors.forename: Lizabeth
authors.initials: L
authors.lastname: Perkins
authors.forename: Norbert
authors.initials: N
authors.lastname: Perrimon
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Mohr
mesh_terms: D000465:Algorithms; D000818:Animals; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism*; D030541:Databases, Genetic; D004331:Drosophila melanogaster / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D015870:Gene Expression; D020407:Internet; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014584:User-Computer Interface
chemical_list: D017931:DNA Primers; D034741:RNA, Small Interfering
keywords: Drosophila; RNAi; gene expression; knockdown evaluation; real-time PCR
references.citation: Biol Proced Online. 2008 Feb 04;10:20-8
references.pmid: 18385808
references.citation: Nucleic Acids Res. 2006 Jan 1;34(Database issue):D684-8
references.pmid: 16381959
references.citation: Genes Dev. 2004 Mar 1;18(5):584-94
references.pmid: 15037551
references.citation: Annu Rev Biochem. 2010;79:37-64
references.pmid: 20367032
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D805-9
references.pmid: 17068075
references.citation: Genetics. 2013 Sep;195(1):37-45
references.pmid: 23792952
references.citation: Dev Dyn. 2005 Mar;232(3):575-84
references.pmid: 15704176
references.citation: Trends Biochem Sci. 2002 Oct;27(10):514-20
references.pmid: 12368087
references.citation: Nucleic Acids Res. 2003 Dec 15;31(24):e154
references.pmid: 14654707
references.citation: J Cell Biol. 2000 Jul 24;150(2):F57-62
references.pmid: 10908587
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D942-5
references.pmid: 18948285
references.citation: Nature. 2000 Mar 16;404(6775):293-6
references.pmid: 10749213
references.citation: Clin Chem. 2009 Apr;55(4):611-22
references.pmid: 19246619
references.citation: Nature. 2007 Jul 12;448(7150):151-6
references.pmid: 17625558
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D715-9
references.pmid: 22067456
references.citation: Genome Biol. 2006;7(7):R61
references.pmid: 16859521
references.citation: Dev Cell. 2002 Nov;3(5):711-22
references.pmid: 12431377
references.citation: Genetics. 2009 Aug;182(4):1089-100
references.pmid: 19487563
references.citation: Development. 2007 Feb;134(4):703-12
references.pmid: 17215303
references.citation: Genetics. 2013 Jan;193(1):51-61
references.pmid: 23105012
references.citation: Genome Biol. 2010;11(6):R61
references.pmid: 20550664
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D1144-9
references.pmid: 22086960
references.citation: Nat Methods. 2011 May;8(5):405-7
references.pmid: 21460824
references.citation: BMC Bioinformatics. 2011 Aug 31;12:357
references.pmid: 21880147
references.citation: Exp Anim. 2010;59(2):125-38
references.pmid: 20484846
references.citation: PLoS One. 2011;6(12):e26182
references.pmid: 22180774
references.citation: Nat Biotechnol. 2008 Mar;26(3):317-25
references.pmid: 18278033
references.citation: Nucleic Acids Res. 2003 Jan 1;31(1):122-3
references.pmid: 12519963
references.citation: Nature. 2011 Mar 24;471(7339):473-9
references.pmid: 21179090
references.citation: Development. 1993 Jun;118(2):401-15
references.pmid: 8223268
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6499-503
references.pmid: 10823906
references.citation: Cold Spring Harb Perspect Biol. 2010 Aug;2(8):a003640
references.pmid: 20534712
references.citation: Genome Res. 2011 Feb;21(2):301-14
references.pmid: 21177962
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D792-9
references.pmid: 19906719