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

Catriona H McIntosh, John Baird, Erich Zinser, Debra J. Woods, Ewan M Campbell, Alan S Bowman

Research output: Contribution to journalArticle

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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.
Original languageEnglish
Pages (from-to)1532-1542
Number of pages11
JournalParasitology
Volume143
Issue number12
Early online date13 Jul 2016
DOIs
Publication statusPublished - Oct 2016

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Keywords

  • ctenocephalides felis
  • normalization
  • RNA
  • gene expression
  • cat flea
  • RNA quality
  • RNA degredation

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