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Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets

Argulus spp. are economically important fish ectoparasites. The development of antiparasitic drugs is thus important and real time PCR is an indispensable tool in drug development. The analytical potential of RT-PCR depends upon accurate normalisation by the use of stable reference genes. Here, we i...

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Autores principales: Sahoo, Pramoda Kumar, Parida, Sonali, Mohapatra, Amruta, Mohanty, Jyotirmaya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892791/
https://www.ncbi.nlm.nih.gov/pubmed/31798003
http://dx.doi.org/10.1038/s41598-019-54881-w
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author Sahoo, Pramoda Kumar
Parida, Sonali
Mohapatra, Amruta
Mohanty, Jyotirmaya
author_facet Sahoo, Pramoda Kumar
Parida, Sonali
Mohapatra, Amruta
Mohanty, Jyotirmaya
author_sort Sahoo, Pramoda Kumar
collection PubMed
description Argulus spp. are economically important fish ectoparasites. The development of antiparasitic drugs is thus important and real time PCR is an indispensable tool in drug development. The analytical potential of RT-PCR depends upon accurate normalisation by the use of stable reference genes. Here, we identified stable reference genes of Argulus siamensis for validation of efficacy of drugs and drug targets. Seven candidate genes were evaluated by evaluating their expression under different states of Argulus using the RefFinder tool. The four algorithms together generated a comprehensive ranking with elongation factor-1 alpha (EF-1α) being the most stable and 18S ribosomal protein (18S) the least stable gene. Taking EF-1α and 18S genes as references, the effectiveness of six anti-parasitic compounds against Argulus was evaluated by studying their effect on the expression pattern of few ion channel genes; this was to understand their mode of action, besides validating the reference genes. EF-1α was found to be the most stable gene in the validation. Collectively, this study is the first report to validate the optimal reference genes of A. siamensis for normalisation, and the potential of the ion channel genes for evaluating effective drug targets in parasite control.
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spelling pubmed-68927912019-12-10 Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets Sahoo, Pramoda Kumar Parida, Sonali Mohapatra, Amruta Mohanty, Jyotirmaya Sci Rep Article Argulus spp. are economically important fish ectoparasites. The development of antiparasitic drugs is thus important and real time PCR is an indispensable tool in drug development. The analytical potential of RT-PCR depends upon accurate normalisation by the use of stable reference genes. Here, we identified stable reference genes of Argulus siamensis for validation of efficacy of drugs and drug targets. Seven candidate genes were evaluated by evaluating their expression under different states of Argulus using the RefFinder tool. The four algorithms together generated a comprehensive ranking with elongation factor-1 alpha (EF-1α) being the most stable and 18S ribosomal protein (18S) the least stable gene. Taking EF-1α and 18S genes as references, the effectiveness of six anti-parasitic compounds against Argulus was evaluated by studying their effect on the expression pattern of few ion channel genes; this was to understand their mode of action, besides validating the reference genes. EF-1α was found to be the most stable gene in the validation. Collectively, this study is the first report to validate the optimal reference genes of A. siamensis for normalisation, and the potential of the ion channel genes for evaluating effective drug targets in parasite control. Nature Publishing Group UK 2019-12-04 /pmc/articles/PMC6892791/ /pubmed/31798003 http://dx.doi.org/10.1038/s41598-019-54881-w Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Sahoo, Pramoda Kumar
Parida, Sonali
Mohapatra, Amruta
Mohanty, Jyotirmaya
Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets
title Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets
title_full Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets
title_fullStr Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets
title_full_unstemmed Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets
title_short Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets
title_sort selection of candidate reference genes for rt-qpcr analysis in argulus siamensis and their validation through screening of drugs and drug targets
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892791/
https://www.ncbi.nlm.nih.gov/pubmed/31798003
http://dx.doi.org/10.1038/s41598-019-54881-w
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