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Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies

Published online by Cambridge University Press:  13 July 2016

CATRIONA H. MCINTOSH ,
JOHN BAIRD ,
ERICH ZINSER ,
DEBRA J. WOODS ,
EWAN M. CAMPBELL  and
ALAN S. BOWMAN
CATRIONA H. MCINTOSH
Affiliation:
Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK
JOHN BAIRD
Affiliation:
Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK
ERICH ZINSER
Affiliation:
Zoetis Inc., 333 Portage Street, Kalamazoo, Michigan 49007, USA
DEBRA J. WOODS
Affiliation:
Zoetis Inc., 333 Portage Street, Kalamazoo, Michigan 49007, USA
EWAN M. CAMPBELL
Affiliation:
Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK
ALAN S. BOWMAN*
Affiliation:
Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK
*
*Corresponding author: School of Biological Sciences (Zoology), University of Aberdeen, Aberdeen, AB24 2TZ, UK. Tel: +44 1224 272877. Fax: +44 1224 272396. E-mail: a.bowman@abdn.ac.uk
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Summary

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.

Keywords

Ctenocephalides felisquantitative real-time PCRnormalizationRNAgene expressioncat fleaRNA qualityRNA degradation
Type
Research Article
Information
Parasitology , Volume 143 , Issue 12 , October 2016 , pp. 1532 - 1542
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Copyright
Copyright © Cambridge University Press 2016 

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