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Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis
Argulosis is one of the most unrestrained economically significant freshwater fish ectoparasitic diseases. Proper selection or normalization of the best reference gene governs the accuracy of results of gene expression studies using real-time PCR. Earlier studies in rohu carp (Labeo rohita) have use...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9932016/ https://www.ncbi.nlm.nih.gov/pubmed/36792637 http://dx.doi.org/10.1038/s41598-023-29325-1 |
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author | Sahoo, Pramoda Kumar Parida, Sonali Parida, Subhadarshini Parida, Priyashree Paul, Anirban |
author_facet | Sahoo, Pramoda Kumar Parida, Sonali Parida, Subhadarshini Parida, Priyashree Paul, Anirban |
author_sort | Sahoo, Pramoda Kumar |
collection | PubMed |
description | Argulosis is one of the most unrestrained economically significant freshwater fish ectoparasitic diseases. Proper selection or normalization of the best reference gene governs the accuracy of results of gene expression studies using real-time PCR. Earlier studies in rohu carp (Labeo rohita) have used reference genes without proper validation. Here, seven candidate reference genes viz., acidic ribosomal protein (ARP0), glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase II (RPo), elongation factor1α (EF1α), α- tubulin (AT), ribosomal protein L 10, and β-actin were evaluated using four algorithms (geNorm, BestKeeper, NormFinder and ∆Ct) followed by a comprehensive gene expression analysis using skin tissue of rohu at varied time points of experimental Argulus siamensis infection. ARP0 and EF1α were found to be the most stable whereas RPo and AT were considered as least stable genes based on basal expression level and variation in expression levels. Validation of candidate reference genes was undertaken by looking into the expression of six immune-related genes using the two most stable and two least stable genes as housekeeping genes in Argulus-infected rohu skin at different time points of infection. An increased expression of immune genes indicated the role of inflammation and the immune modulation process at the site of attachment of parasites in governing infection. |
format | Online Article Text |
id | pubmed-9932016 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-99320162023-02-17 Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis Sahoo, Pramoda Kumar Parida, Sonali Parida, Subhadarshini Parida, Priyashree Paul, Anirban Sci Rep Article Argulosis is one of the most unrestrained economically significant freshwater fish ectoparasitic diseases. Proper selection or normalization of the best reference gene governs the accuracy of results of gene expression studies using real-time PCR. Earlier studies in rohu carp (Labeo rohita) have used reference genes without proper validation. Here, seven candidate reference genes viz., acidic ribosomal protein (ARP0), glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase II (RPo), elongation factor1α (EF1α), α- tubulin (AT), ribosomal protein L 10, and β-actin were evaluated using four algorithms (geNorm, BestKeeper, NormFinder and ∆Ct) followed by a comprehensive gene expression analysis using skin tissue of rohu at varied time points of experimental Argulus siamensis infection. ARP0 and EF1α were found to be the most stable whereas RPo and AT were considered as least stable genes based on basal expression level and variation in expression levels. Validation of candidate reference genes was undertaken by looking into the expression of six immune-related genes using the two most stable and two least stable genes as housekeeping genes in Argulus-infected rohu skin at different time points of infection. An increased expression of immune genes indicated the role of inflammation and the immune modulation process at the site of attachment of parasites in governing infection. Nature Publishing Group UK 2023-02-15 /pmc/articles/PMC9932016/ /pubmed/36792637 http://dx.doi.org/10.1038/s41598-023-29325-1 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Sahoo, Pramoda Kumar Parida, Sonali Parida, Subhadarshini Parida, Priyashree Paul, Anirban Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis |
title | Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis |
title_full | Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis |
title_fullStr | Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis |
title_full_unstemmed | Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis |
title_short | Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis |
title_sort | stability evaluation and validation of appropriate reference genes for real-time pcr expression analysis of immune genes in the rohu (labeo rohita) skin following argulosis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9932016/ https://www.ncbi.nlm.nih.gov/pubmed/36792637 http://dx.doi.org/10.1038/s41598-023-29325-1 |
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