Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris

Sirakov, M; Zarrella, Ilaria; Borra, Marco; Rizzo, Francesca; Biffali, Elio; Arnone, Maria Ina; Fiorito, Graziano

doi:10.1186/1471-2199-10-70

Cited by 30 publications

(29 citation statements)

References 24 publications

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“…According to our results, UBI is a suitable reference for measuring the gene expression in both paralarvae sample sets. This result agrees with that of Sirakov et al (2009) who identified UBI as an appropriate reference gene to analyze the gene expression in octopus brain samples. Therefore, the gene UBI seems to be the best option as a stable gene for measuring the gene expression in O.…”

Section: Discussionsupporting

confidence: 93%

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

García-Fernández

Castellanos-Martínez

Iglesias

et al. 2016

Journal of Invertebrate Pathology

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The 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 34days) and paralarvae of 20days 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.

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Section: Discussionsupporting

confidence: 93%

“…β-actin and elongation factor 1 alpha primers used in brain of O. vulgaris (Sirakov et al 2009) were also included in the analysis to test their validity for octopus paralarvae. Specificity of amplification was confirmed by melting curve and agarose gel electrophoresis.…”

Section: Primers Design and Rt-qpcrmentioning

confidence: 99%

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

García-Fernández

Castellanos-Martínez

Iglesias

et al. 2016

Journal of Invertebrate Pathology

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“…Ribosomal RNA genes (i.e., 18S) were not included as potential RG candidates in various studies because they may be related to the physiological and/or metabolic status of the analyzed organism (Sirakov et al 2009) or because they are not polyadenylated upon transcription and cannot be utilized when using polydT priming in the reversetranscription reaction (Ransbotyn and Reusch 2006). In our analyses, retro-transcription kit with both polydT and random primers has been used, and 18S was assigned as best RG by BestKeeper in all the tested conditions (also for the universal RG assessment analyses), except for depth variations.…”

Section: Discussionmentioning

confidence: 99%

“…For example, ACT, EF1A and 18S were the best RGs in chicory (Cichorium intybus) in leaf and root samples (Maroufi et al 2010), EF1A and UBI in cucumber tissues treated with hormones and abiotic stress (Wan et al 2010), NTUBC2, L25 and EF1A in developmentally distinct tissues or tissues exposed to abiotic stress in tobacco (Nicotiana tobacum) (Schmidt and Delaney 2010) and TBP was between the best RGs in Zostera marina after temperature treatments (Ransbotyn and Reusch 2006). TUB and UBI were used as RGs for different Octopus vulgaris tissues (Sirakov et al 2009), UBC, GAPDH and ACT in Daphnia magna exposed to ibuprofen (Heckmann et al 2006), GAPDH in the copepod Calanus helgolandicus exposed to a toxic diet (Lauritano et al 2011), and ciclofillin was among the putative RGs for both the seagrass Zostera marina after temperature treatments (Ransbotyn and Reusch 2006), and the Cladoceran Daphnia magna exposed to ibuprofen (Heckmann et al 2006). In order to compare the stability of reference genes, four genes of interest, two taken from the ones differentially expressed at different depths (P. oceanica SSH library; Procaccini et al 2010) and two putatively involved in the response to salinity and pH, were also selected ( Table 2).…”

Section: Selection Of Genes and Primer Designmentioning

confidence: 99%

Reference genes assessment for the seagrass Posidonia oceanica in different salinity, pH and light conditions

et al. 2012

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The stability on the expression level of putative reference genes (RGs) for reverse transcriptase-quantitative PCR (RT-qPCR) was tested in the seagrass Posidonia oceanica for plants collected in different environmental conditions (at different depths and in different salinity and pH). Besides adult plants, seedlings were also used for pH and salinity experiments. The putative RGs encompassed the most frequently used in other species to normalize RT-qPCR in a range of experimental conditions. Assignment of the best RGs has been performed with three Excelbased applets, BestKeeper, geNorm and NormFinder, and the results showed that best RGs may change depending on the experimental conditions tested, plant age and on the software utilized. Preferred sets of RGs are proposed for each of the conditions and a general procedure concerning their selection is discussed. In addition, expression levels of four genes involved in plant response to light, salinity and pH variation have also been evaluated.

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“…Therefore, we aimed to develop and test a new set of endogenous genes as controls, for use in our study, but also for subsequent use by the wider community, just as has been the case in some other species (Hibbeler, Scharsack & Becker, 2008; Li et al , 2017; Olias et al , 2014; Sirakov et al , 2009).…”

Section: Introductionmentioning

confidence: 99%

A set of endogenous control genes for use in quantitative real-time PCR experiments reveal that the wild-type formin Ldia2 is enriched in the early pond snail embryo

Johnson¹,

Davison²

2019

Preprint

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Although the pond snail Lymnaea stagnalis is an emerging model organism for molecular studies in a wide variety of fields including development, biomineralisation and neurophysiology, there are a limited number of verified endogenous control genes for use in quantitative real-time PCR (qRT-PCR). As part of larger study on snail chirality or left-right asymmetry, we wished to assay relative gene expression in pond snail embryos, so we evaluated six new candidate control genes, by comparing their expression in three tissues (ovotestis, foot, and embryo) and across three programs (geNorm, Normfinder and Bestkeeper). The specific utility of these control genes was then tested by investigating the relative expression of six experimental transcripts, including the formin Ldia2, a gene that has been associated with chirality in L. stagnalis. All six control genes were found to be suitable for use. Of the six experimental genes that were tested, it was found that all were relatively depleted in the early embryo compared with other tissues, except the formin gene Ldia2. Instead, transcripts of the wild type Ldia2dex were enriched in the embryo, whereas a non-functional frameshifted version Ldia2sin was severely depleted. These differences in Ldia2sin expression were less evident in the ovotestis and not evident in the foot tissue, suggesting that nonsense-mediated decay may be obscured in actively transcribing tissues. This work therefore provides a set of control genes that may be useful to the wider community, and shows how they may be used to assay differences in expression in the early embryo.

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Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris

Cited by 30 publications

References 24 publications

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

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

Reference genes assessment for the seagrass Posidonia oceanica in different salinity, pH and light conditions

A set of endogenous control genes for use in quantitative real-time PCR experiments reveal that the wild-type formin Ldia2 is enriched in the early pond snail embryo

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