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



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.


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*Corresponding author: School of Biological Sciences (Zoology), University of Aberdeen, Aberdeen, AB24 2TZ, UK. Tel: +44 1224 272877. Fax: +44 1224 272396. E-mail:


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Andersen, C. L., Jensen, J. L. and Orntoft, T. F. (2004). Normalization of real-time quantitative reverse transcription-PCR data: a mode-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research 64, 52455250.
Beugnet, F., Bourdeau, P., Chalvet-Monfray, K., Cozma, V., Farkas, R., Guillot, J., Halos, L., Joachim, A., Losson, B., Miro, G., Otranto, D., Renaud, M. and Rinaldi, L. (2014). Parasites of domestic cats in Europe: co-infestations and risk factors. Parasites and Vectors 7, 291.
Boda, E., Pini, A., Hoxha, E., Parolisi, R. and Tempia, F. (2008). Selection of reference genes for quantitative real-time RT-PCR studies in mouse brain. Journal of Molecular Neuroscience 37, 238253.
Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandescompele, J. and Wittwer, C. T. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55, 611622.
Chen, Y., Evans, J., Hamilton, M. and Feldlaufer, M. (2007). The influence of RNA integrity on the detection of honey bee viruses: molecular assessment of different sample storage methods. Journal of Apicultural Research 46, 8187.
Derveaux, S., Vandesompele, J. and Hellemans, J. (2010). How to do successful gene expression analysis using real-time PCR. Methods 50, 227230.
Dos Santos, G., Schroeder, A. J., Goodman, J. L., Strelets, V. B., Crosby, M. A., Thurmond, J., Emmert, D. B. and Gelbart, W. M. (2015). FlyBase: introduction of the Drosophila melanogaster release 6 reference genome assembly and large-scale migration of genome annotations. Nucleic Acids Research 43, D690D697.
Dreher-Lesnick, S. M., Ceraul, S. M., Lesnick, S. C., Gillespie, J. J., Anderson, J. M., Jochim, R. C., Valenzuela, J. G. and Azad, A. F. (2010). Analysis of Rickettsia typhi-infected and uninfected cat flea (Ctenocephalides felis) midgut cDNA libraries: deciphering molecular pathways involved in host response to R. typhi infection. Insect Molecular Biology 19, 229241.
Gaines, P. J., Brandt, K. S., Eisele, A. M., Wagner, W. P., Bozic, C. M. and Wisnewski, N. (2002). Analysis of expressed sequence tags from subtracted and unsubtracted Ctenocephalides felis hindgut and Malpighian tubule cDNA libraries. Insect Molecular Biology 11, 299306.
Greene, W. K., Macnish, M. G., Rice, K. L. and Thompson, R. C. A. (2015). Identification of genes associated with blood feeding in the cat flea, Ctenocephalides felis . Parasites and Vectors 8, 368.
Henry, K. M., Jiang, J., Rozmajzl, P. J., Azad, A. F., Macaluso, K. R. and Richards, A. L. (2007). Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever. Molecular and Cellular Probes 21, 1723.
Huggett, J., Dheda, K., Bustin, S. and Zumla, A. (2005). Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity 6, 279284.
Kernif, T., Stafford, K., Coles, G. C., Bitam, I., Papa, K., Chiaroni, J., Raoult, D. and Parola, P. (2015). Responses of artificially reared cat fleas Ctenocephalides felis felis (Bouche, 1835) to different mammalian bloods. Medical and Veterinary Entomology 29, 171177.
Kidd, M., Nadler, B., Mane, S., Eick, G., Malfertheiner, M., Champaneria, M., Pfragner, R. and Modlin, I. (2007). GeneChip, geNorm, and gastrointestinal tumors: novel reference genes for real-time PCR. Physiological Genomics 30, 363370.
Kosir, R., Acimovic, J., Golicnik, M., Perse, M., Majdic, G., Fink, M. and Rozman, D. (2010). Determination of reference genes for circadian studies in different tissues and mouse strains. BMC Molecular Biology 11, 60.
Li, R., Wang, S., Wu, Q., Yang, N., Yang, X., Pan, H., Zhou, X., Bai, L., Xu, B., Zhou, X. and Zhang, Y. (2013). Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). PLoS ONE 8, e53006.
Misof, B., Liu, S., Meusemann, K., Peters, R. S., Donath, A., Mayer, C., Frandsen, P. B., Ware, J., Flouri, T., Beutel, R. G., Niehuis, O., Petersen, M., Izquierdo-Carrasco, F., Wappler, T., Rust, J., Aberer, A. J., Aspöck, U., Aspöck, H., Bartel, D., Blanke, A., Berger, S., Böhm, A., Buckley, T. R., Calcott, B., Chen, J., Friedrich, F., Fukui, M., Fujita, M., Greve, C., Grobe, P., Gu, S. et al. (2014). Phylogenomics resolves the timing and pattern of insect evolution. Science 346, 763767.
Odhiambo, A. M., Maina, A. N., Taylor, M. L., Jiang, J. and Richards, A. L. (2014). Development and validation of a quantitative real-time polymerase chain reaction assay specific for the detection of Rickettsia felis and not Rickettsia felis-like organisms. Vector-Borne and Zoonotic Diseases 14, 476481.
Omondi, B. A., Latorre-Estivalis, J. M., Oliveira, I. H. R., Ignell, R. and Lorenzo, M. G. (2015). Evaluation of reference genes for insect olfaction studies. Parasites and Vectors 8, 243.
Pan, M. L., Bell, W. J. and Telfer, W. H. (1969). Vitellogenic blood protein synthesis by insect fat body. Science 165, 393394.
Pfaffl, M. W., Tichopad, A., Prgomet, C. and Neuvians, T. P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnology Letters 26, 509515.
Ribeiro, J. M. C., Assumpção, T. C. F., Ma, D., Alvarenga, P. H., Pham, V. M., Andersen, J. F., Francischetti, I. M. B. and Macaluso, K. R. (2012). An insight into the sialotranscriptome of the cat flea, Ctenocephalides felis . PLoS ONE 7, e44612.
Rust, M. K. and Dryden, M. W. (1997). The biology, ecology, and management of the cat flea. Annual Review of Entomology 42, 451473.
Scharlaken, B., de Graaf, D., Goossens, K., Brunain, M., Peelman, L. J. and Jacobs, F. J. (2008). Reference gene selection for insect expression studies using quantitative real-time PCR: the head of the honeybee, Apis mellifera, after a bacterial challenge. Journal of Insect Science 8, 33.
Shakeel, M., Zhu, X., Kang, T., Wan, H. and Li, J. (2015). Selection and evaluation of reference genes for quantitative gene expression studies in cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). Journal of Asia-Pacific Entomology 18, 123130.
Tan, Q. Q., Zhu, L., Li, Y., Liu, W., Ma, W. H., Lei, C. L. and Wang, X. P. (2015). A de Novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi . PLoS ONE 10, e0118693.
Teng, X., Zhang, Z., He, G., Yang, L. and Li, F. (2012). Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects. Journal of Insect Science 12, 60.
Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., Grisar, T., Igout, A. and Heinen, E. (1999). Housekeeping genes as internal standards: use and limits. Journal of Biotechnology 75, 291295.
Traversa, D. (2013). Fleas infesting pets in the era of emerging extra-intestinal nematodes. Parasites and Vectors 6, 59.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. and Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3, RESEARCH0034.1
Winnebeck, E. C., Millar, C. D. and Warman, G. R. (2010). Why does insect RNA look degraded? Journal of Insect Science 10, 159.
Zhai, Y., Lin, Q., Zhou, X., Zhang, X., Liu, T. and Yu, Y. (2014). Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera: Drosophilidae). PloS ONE 9, e106800.


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



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