title: Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies.
abstract: The cat flea, Ctenocephalides felis, is a major pest species on companion animals thus of significant importance to the animal health industry. The aim of this study was to develop sampling and storage protocols and identify stable reference genes for gene expression studies to fully utilize the growing body of molecular knowledge of C. felis. RNA integrity was assessed in adult and larvae samples, which were either pierced or not pierced and stored in RNAlater at ambient temperature. RNA quality was maintained best in pierced samples, with negligible degradation evident after 10 days. RNA quality from non-pierced samples was poor within 3 days. Ten candidate reference genes were evaluated for their stability across four group comparisons (developmental stages, genders, feeding statuses and insecticide-treatment statuses). Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), 60S ribosomal protein L19 (RPL19) and elongation factor-1α (Ef) were ranked highly in all stability comparisons, thus are recommended as reference genes under similar conditions. Employing just two of these three stable reference genes was sufficient for accurate normalization. Our results make a significant contribution to the future of gene expression studies in C. felis, describing validated sample preparation procedures and reference genes for use in this common pest.
authors.affiliation: Institute of Biological and Environmental Sciences, University of Aberdeen,Tillydrone Avenue,Aberdeen AB24 2TZ,UK.
authors.forename: Catriona H
authors.initials: CH
authors.lastname: McIntosh
authors.affiliation: Institute of Biological and Environmental Sciences, University of Aberdeen,Tillydrone Avenue,Aberdeen AB24 2TZ,UK.
authors.forename: John
authors.initials: J
authors.lastname: Baird
authors.affiliation: Zoetis Inc.,333 Portage Street,Kalamazoo,Michigan 49007,USA.
authors.forename: Erich
authors.initials: E
authors.lastname: Zinser
authors.affiliation: Zoetis Inc.,333 Portage Street,Kalamazoo,Michigan 49007,USA.
authors.forename: Debra J
authors.initials: DJ
authors.lastname: Woods
authors.affiliation: Institute of Biological and Environmental Sciences, University of Aberdeen,Tillydrone Avenue,Aberdeen AB24 2TZ,UK.
authors.forename: Ewan M
authors.initials: EM
authors.lastname: Campbell
authors.affiliation: Institute of Biological and Environmental Sciences, University of Aberdeen,Tillydrone Avenue,Aberdeen AB24 2TZ,UK.
authors.forename: Alan S
authors.initials: AS
authors.lastname: Bowman
mesh_terms: D000818:Animals; D058251:Ctenocephalides / Q000235:genetics*; D004772:Entomology / Q000379:methods* / Q000592:standards; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D011309:Preservation, Biological / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D012015:Reference Standards
chemical_list: D012313:RNA
keywords: Ctenocephalides felis; RNA; RNA degradation; RNA quality; cat flea; gene expression; normalization; quantitative real-time PCR