Skip to main content Accessibility help
×
Home
Hostname: page-component-684bc48f8b-mkrr2 Total loading time: 0.264 Render date: 2021-04-11T23:06:50.631Z Has data issue: true Feature Flags: { "shouldUseShareProductTool": true, "shouldUseHypothesis": true, "isUnsiloEnabled": true, "metricsAbstractViews": false, "figures": false, "newCiteModal": false, "newCitedByModal": true }

Article contents

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
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
E-mail address:

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.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2016 

Access options

Get access to the full version of this content by using one of the access options below.

References

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.CrossRefGoogle Scholar
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle Scholar
Derveaux, S., Vandesompele, J. and Hellemans, J. (2010). How to do successful gene expression analysis using real-time PCR. Methods 50, 227230.CrossRefGoogle Scholar
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
Huggett, J., Dheda, K., Bustin, S. and Zumla, A. (2005). Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity 6, 279284.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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.Google Scholar
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle Scholar
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.CrossRefGoogle ScholarPubMed
Pan, M. L., Bell, W. J. and Telfer, W. H. (1969). Vitellogenic blood protein synthesis by insect fat body. Science 165, 393394.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
Rust, M. K. and Dryden, M. W. (1997). The biology, ecology, and management of the cat flea. Annual Review of Entomology 42, 451473.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle Scholar
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.CrossRefGoogle Scholar
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.Google Scholar
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.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
Traversa, D. (2013). Fleas infesting pets in the era of emerging extra-intestinal nematodes. Parasites and Vectors 6, 59.CrossRefGoogle ScholarPubMed
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, RESEARCH0034.1CrossRefGoogle ScholarPubMed
Winnebeck, E. C., Millar, C. D. and Warman, G. R. (2010). Why does insect RNA look degraded? Journal of Insect Science 10, 159.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle Scholar

Altmetric attention score

Full text views

Full text views reflects PDF downloads, PDFs sent to Google Drive, Dropbox and Kindle and HTML full text views.

Total number of HTML views: 54
Total number of PDF views: 149 *
View data table for this chart

* Views captured on Cambridge Core between September 2016 - 11th April 2021. This data will be updated every 24 hours.

Send article to Kindle

To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

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

Send article to Dropbox

To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

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

Send article to Google Drive

To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

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

Reply to: Submit a response


Your details


Conflicting interests

Do you have any conflicting interests? *