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Title

Selection of reliable reference genes for RT-qPCR studies in Octopus vulgaris paralarvae during development and immune-stimulation

AuthorsGarcía-Fernández, Pablo; Castellanos-Martínez, Sheila ; Iglesias, José; Otero, J. J.; Gestal, C.
KeywordsOctopus vulgaris
Paralarvae
Reference gene
RT-qPCR
Development
Infection
Issue Date2016
PublisherElsevier
CitationJournal of Invertebrate Pathology 138: 57-62 (2016)
AbstractThe common octopus, Octopus vulgaris is a new candidate species for aquaculture. However, rearing of octopus paralarvae is hampered by high mortality and poor growth rates that impede its entire culture. The study of genes involved in the octopus development and immune response capability could help to understand the key of paralarvae survival and thus, to complete the octopus life cycle. Quantitative real-time PCR (RT-qPCR) is the most frequently tool used to quantify the gene expression because of specificity and sensitivity. However, reliability of RT-qPCR requires the selection of appropriate normalization genes whose expression must be stable across the different experimental conditions of the study. Hence, the aim of the present work is to evaluate the stability of six candidate genes: β-actin (ACT), elongation factor 1-α (EF), ubiquitin (UBI), β-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GADPH) and ribosomal RNA 18 (18S) in order to select the best reference gene. The stability of gene expression was analyzed using geNorm, NormFinder and Bestkeeper, in octopus paralarvae of seven developmental stages (embryo, paralarvae of 0, 10, 15, 20, 30 and 34 days) and paralarvae of 20 days after challenge with Vibrio lentus and Vibrio splendidus. The results were validated by measuring the expression of PGRP, a stimuli-specific gene. Our results showed UBI, EF and 18S as the most suitable reference genes during development of octopus paralarvae, and UBI, ACT and 18S for bacterial infection. These results provide a basis for further studies exploring molecular mechanism of their development and innate immune defense
Description6 pages, 3 figures, 2 tables
Publisher version (URL)https://doi.org/10.1016/j.jip.2016.06.003
URIhttp://hdl.handle.net/10261/192579
DOI10.1016/j.jip.2016.06.003
ISSN0022-2011
E-ISSN1096-0805
Appears in Collections:(IIM) Artículos
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