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Evaluation of candidate reference genes for quantitative RTqPCR analysis in goldfish (Carassius auratus L.) in healthy and CyHV-2 infected fish

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Title Evaluation of candidate reference genes for quantitative RTqPCR analysis in goldfish (Carassius auratus L.) in healthy and CyHV-2 infected fish
Not Available
 
Creator Arathi Dharmaratnam
Arun Sudhagar
Sundar Raj N
Sweta Das
Swaminathan T.R.
 
Subject Goldfish
CyHV-2
Reference gene
Real time PCR
Expression stability
 
Description We developed a set of genes that can be reliable in the normalization of RTqPCR gene expression data in goldfish and provides information on the stable endogenous gene to be used in gene expression profiling among healthy and CyHV-2 infected fish.
The accuracy of quantitative real time PCR (RTqPCR) can be attained only when a suitable reference gene is
used. The gene expression for a particular gene may vary within different cells at different conditions. Hence, the suitability and stability of various potential reference genes have to be determined for expression studies. In this study, we have examined the potential of four different reference genes including β-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3P-dehydrogenase (GAPDH), and elongation factor 1 alpha (EF1AA) in seven different tissues including gill, liver, kidney, spleen, heart, muscle and intestine of goldfish (Carassius auratus). The housekeeping genes were analyzed from healthy fish and in CyHV-2 challenged fish. Based upon the real time PCR results the gene expression varied among the genes and in tissues. The expression levels of the housekeeping genes were then compared and evaluated with the RefFinder web tool which analyses results using four different algorithms – BestKeeper, delta Ct, geNorm and NormFinder. EF1AA was ranked to be the best gene in healthy fish by BestKeeper and geNorm analysis. The delta Ct and NormFinder algorithm have found 18S to be a stable gene in healthy fish but 18S was given to be least expressed in challenged fish. ACTB was also given as a stable gene by geNorm analysis in both healthy and challenged fish. Also, in CyHV-2 challenged fish, EF1AA was identified as the best gene by all the three analysis except by BestKeeper analysis, where it has ranked GADPH as the best housekeeping gene. Expression of the four candidate reference genes differed across all tissue types tested, inferring that a thorough study of the reference genes is necessary for cross tissue comparison. These results can be further used in the immune gene response study of goldfish infected with any viral pathogen to develop better health strategies in the disease management of goldfish aquaculture.
ICAR National Fellow Scheme of ICAR (ICAR letter no F.No Agri.edn.27/ 2/2015 HRD dated 13.02.2017), Department of Agricultural Research and Education, Ministry of Agriculture and Farmer’s Welfare, Government of India.
 
Date 2024-05-02T17:07:05Z
2024-05-02T17:07:05Z
2021-05-15
 
Type Research Paper
 
Identifier Arathi Dharmaratnam, Arun Sudhagar, Sundar Raj Nithianantham, Sweta Das, Thangaraj Raja Swaminathan, Evaluation of candidate reference genes for quantitative RTqPCR analysis in goldfish (Carassius auratus L.) in healthy and CyHV-2 infected fish, Veterinary Immunology and Immunopathology, Volume 237, 2021, 110270, ISSN 0165-2427, https://doi.org/10.1016/j.vetimm.2021.110270.
0165-2427
http://krishi.icar.gov.in/jspui/handle/123456789/82577
 
Language English
 
Relation Not Available;
 
Publisher Elsevier