Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp

Leelatanawit, Rungnapa; Klanchui, Amornpan; Uawisetwathana, Umaporn; Karoonuthaisiri, Nitsara

doi:10.1371/journal.pone.0052677

Cited by 37 publications

(32 citation statements)

References 68 publications

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“…Amounts of prostanoid biosynthesis gene transcripts relative to that of the house-keeping gene elongation factor 1α (EF1α) were obtained using the standard curve method [55]. The specificity of the PCR product was confirmed by agarose gel electrophoresis and melting curve analysis performed from 55 °C to 95 °C with a continuous fluorescent reading at 0.5 °C increments.…”

Section: Methodsmentioning

confidence: 99%

Insights into the Prostanoid Pathway in the Ovary Development of the Penaeid Shrimp Penaeus monodon

et al. 2013

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The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp Penaeus monodon based on the amplification of nine prostanoid biosynthesis genes: cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase, thromboxane A synthase and cyclooxygenase. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F2α (PGF2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in P. monodon ovaries along with the previously identified PUFAs and prostaglandin E2 (PGE2) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using in vitro activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of prostaglandin F synthase gene transcripts and PGF2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF2α biosynthesis in shrimp ovarian development.

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Section: Methodsmentioning

confidence: 99%

Insights into the Prostanoid Pathway in the Ovary Development of the Penaeid Shrimp Penaeus monodon

et al. 2013

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“…The results presented are from three independent ( n = 3) biological replicates, and statistical significance was determined by an unpaired two‐tailed Student's t ‐test. Data were normalized to the YlActin ( Yl β ‐Actin ) and LvActin ( Lv β ‐Actin ) reference genes (Table ) respectively (Leelatanawit, Klanchui, Uawisetwathana & Karoonuthaisiri, ; Mansour et al., ). The method used to analyse the data from real‐time PCR experiments corresponds to the relative quantification method, or

2^{- normalΔ normalΔ C_{T}}

method, where the ΔΔ C T value =

(false(C_{{normalT}_{1 Target}} - C_{{normalT}_{1 Reference}} false) - false(C_{{normalT}_{0 Target}} - C_{{normalT}_{0 Reference}} false))

(Livak & Schmittgen, ).…”

Section: Methodsmentioning

confidence: 99%

Production of specific dsRNA against white spot syndrome virus in the yeastYarrowia lipolytica

et al. 2017

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White spot syndrome virus (WSSV) is the most aggressive disease affecting cultured shrimp. One possibility to tackle it is by means of RNA interference (RNAi) induced by the presence of double-stranded RNA (dsRNA). Normally, dsRNA is a product of the cellular machinery to gene regulation, but it can be produced synthetically and introduced into specific tissues or cells and thereby induce RNAi. Although in vitro production of dsRNA is possible, this is high cost. An alternative is to produce dsRNA in vivo using biological systems such as bacteria or yeasts. In this regard, Yarrowia lipolytica offers distinctive advantages for dsRNA production. The objective was to develop a Y. lipolytica strain able to produce dsRNA-specific against WSSV and to evaluate its antiviral activity in the white leg shrimp Litopenaeus vannamei.From the 0.4 and 0.6 Kb fragments of the ORF89 gene, a dsRNA-ORF89-producing construct was built in the plasmid pJC410; the resulting construct (pARY410) was used to transform Y. lipolytica to drive the specific expression of dsRNA-ORF89.Yeast colonies positive to the WSSV-ORF89 gene were selected. The expression of dsRNA-ORF89 and RNAse III was measured being detected at 32 and 48 hr. Subsequently, the antiviral activity of dsRNA-ORF89 was tested in a WSSV challenge bioassay. The results showed survival in dsRNA-ORF89 shrimp (25%) compared to control organisms treated with total RNA from the yeast P01-AS harvested at 32 hr. In conclusion, Y. lipolytica is a convenient host to produce and deliver dsRNA-ORF89 able to protect WSSV-challenged shrimp. K E Y W O R D Santiviral, Litopenaeus vannamei, ORF89, RNAi, RNAse III

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“…Relative quantification of gene expression has been accepted as the gold standard method in several modern techniques such as quantitative real-time PCR, microarray, and high-throughput sequencing (Grozinger et al, 2007; Wang et al, 2012a; Wang et al, 2012b; Paria et al, 2013; Das et al, 2015; Van Hoeck et al, 2015) whose accuracy depends largely on the stability of the reference gene employed for normalization (Bustin, 2002; Leelatanawit et al, 2012). Ideally, to qualify as stable, a reference gene should express at a constant level in each sample across the groups/categories (treatments, tissues, developmental stages, etc).…”

Section: Introductionmentioning

confidence: 99%

“…However their precision reduces in a small panel comprised of reference genes of divergent nature (Silver et al, 2006). It is always advised to evaluate a large number of reference genes and use at least two for the normalization of a target gene (Vandesompele et al, 2002; Andersen et al, 2004; Silver et al, 2006; Chandna et al, 2012; Leelatanawit et al, 2012; Xie et al, 2012; Ling et al, 2014; Taki and Zhang, 2013; Guo et al, 2014; Hildyard and Wells, 2014). Selecting and employing more than one reference gene overburdens a researcher with limited funds and reference gene sequence information.…”

Section: Introductionmentioning

confidence: 99%

Analysis of variance, normal quantile-quantile correlation and effective expression support of pooled expression ratio of reference genes for defining expression stability

Priyadarshi¹,

Das

Kumar

et al. 2017

Heliyon

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Identification of a reference gene unaffected by the experimental conditions is obligatory for accurate measurement of gene expression through relative quantification. Most existing methods directly analyze variability in crossing point (Cp) values of reference genes and fail to account for template-independent factors that affect Cp values in their estimates. We describe the use of three simple statistical methods namely analysis of variance (ANOVA), normal quantile-quantile correlation (NQQC) and effective expression support (EES), on pooled expression ratios of reference genes in a panel to overcome this issue. The pooling of expression ratios across the genes in the panel nullify the sample specific effects uniformly affecting all genes that are falsely reflected as instability. Our methods also offer the flexibility to include sample specific PCR efficiencies in estimations, when available, for improved accuracy. Additionally, we describe a correction factor from the ANOVA method to correct the relative fold change of a target gene if no truly stable reference gene could be found in the analyzed panel. The analysis is described on a synthetic data set to simplify the explanation of the statistical treatment of data.

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Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp

Cited by 37 publications

References 68 publications

Insights into the Prostanoid Pathway in the Ovary Development of the Penaeid Shrimp Penaeus monodon

Insights into the Prostanoid Pathway in the Ovary Development of the Penaeid Shrimp Penaeus monodon

Production of specific dsRNA against white spot syndrome virus in the yeastYarrowia lipolytica

Analysis of variance, normal quantile-quantile correlation and effective expression support of pooled expression ratio of reference genes for defining expression stability

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