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

Arathi Dharmaratnam; Arun Sudhagar; Sundar Raj Nithianantham; Sweta Das; Thangaraj Raja Swaminathan

doi:10.1016/j.vetimm.2021.110270

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

Vet Immunol Immunopathol. 2021 Jul:237:110270. doi: 10.1016/j.vetimm.2021.110270. Epub 2021 May 15.

Authors

Arathi Dharmaratnam¹, Arun Sudhagar², Sundar Raj Nithianantham², Sweta Das², Thangaraj Raja Swaminathan³

Affiliations

¹ Peninsular and Marine Fish Genetic Resources Centre, ICAR- National Bureau of Fish Genetic Resources, ICAR CMFRI Campus, Kochi - 682 018, Kerala, India. Electronic address: arathid022@gmail.com.
² Peninsular and Marine Fish Genetic Resources Centre, ICAR- National Bureau of Fish Genetic Resources, ICAR CMFRI Campus, Kochi - 682 018, Kerala, India.
³ Peninsular and Marine Fish Genetic Resources Centre, ICAR- National Bureau of Fish Genetic Resources, ICAR CMFRI Campus, Kochi - 682 018, Kerala, India. Electronic address: rajanbfgr@gmail.com.

PMID: 34015681
DOI: 10.1016/j.vetimm.2021.110270

Abstract

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 (EF1A_A) 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. EF1A_A 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, EF1A_A 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.

Keywords: CyHV-2; Expression stability; Goldfish; Real time PCR; Reference gene.

MeSH terms

Animals
Fish Diseases / genetics*
Genes, Essential*
Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
Goldfish / genetics*
Herpesviridae Infections / genetics
Herpesviridae Infections / veterinary*
Herpesviridae*
Organ Specificity
Real-Time Polymerase Chain Reaction / veterinary*
Reference Values

Substances

Glyceraldehyde-3-Phosphate Dehydrogenases

Supplementary concepts

Cyprinid herpesvirus 2