Selection and validation of candidate reference genes for quantitative real-time PCR studies in the shrimp Penaeus vannamei under viral infection

Validation of reference genes for gene expression analysis in melanin‐injected black tiger shrimp ( Penaeus monodon )

“…The selection and validation of reference genes for evaluating the expression of target genes by RT-qPCR have been studied in different organisms, including shrimp (Alvarez- Lee et al, 2020;Chapuis et al, 2011;Ingerslev et al, 2006;Jorgensen et al, 2006;Leelatanawit et al, 2012;Liman et al, 2013;Pan et al, 2015;Valenzuela-Castillo et al, 2017;Wang et al, 2015;Yang et al, 2016).…”

“…Although there are studies of RT‐qPCR using a single reference gene, the authors must present clear evidence of the stability of expression of this reference gene under the experimental conditions described. To date, the ΔC T method, geNorm, NormFinder, BestKeeper and the integrated tool RefFinder algorithms have been most commonly used for the evaluation of reference gene expression stability (Alvarez‐Lee et al, 2020; Hu et al, 2018; Leelatanawit et al, 2012; Pan et al, 2015; Valenzuela‐Castillo et al, 2017; Wang et al, 2015).…”

“…EF1‐α was validated as the most reliable internal control gene for qPCR gene expression analysis of the reproductive system in black tiger shrimp ( P. monodon ) (Leelatanawit et al, 2012). In a study of whiteleg shrimp ( Litopenaeus vannamei ) under viral infection, Valenzuela‐Castillo et al (2017) showed that GAPDH was the most suitable reference gene to normalize gene expression data from Penstyldensovirus 1 challenge over 21 days, while EF1‐α , β‐tubulin , and ribosome protein L8 encoding gene ( RPL8 ) were identified as unstably expressed during the infection process. In another study, ribosome protein L18 encoding gene ( RPL18 ) was the most suitable reference gene for adult Macrobrachium nipponense tissues, while EF1‐α was the most stable in different ovarian and embryo stages and in WSSV infection, and β‐actin was the most stable reference gene under hypoxic stress (Hu et al, 2018).…”

Selection of reference genes for the study of relative gene expression during development of Litopenaeus vannamei larvae

“…Metabolic enzymes such as GAPDH and PK participate in glycolysis, although they perform other roles (Jung et al, 2012). GAPDH has been reported to be a good reference gene in experimental assays using adult shrimp (Dhar et al, 2009; Valenzuela‐Castillo et al, 2017), however, it was not the best option for measuring gene expression in embryonic stages of M. olfersii (Jaramillo et al, 2017). This could be related to the metabolic changes occurring in metamorphic stages; for instance, during the inter‐molting stage, glycolytic pathways are active, however, these pathways stop when animals enter to the pre‐molting stage, depleting the GAPDH protein requirement (Gao et al, 2017 ) .…”

“…Reverse transcription coupled with quantitative real time polymerase chain reaction (RT‐qPCR) is the most popular method to calculate relative gene expression under different biological contexts (Kozera & Rapacz, 2013; Valenzuela‐Castillo, Mendoza‐Cano, Enríquez‐Espinosa, Grijalva‐Chon, & Sánchez‐Paz, 2017). Accuracy of this method depends on stable reference genes to normalize gene expression, since the amount of mRNA may show sample‐to‐sample variations, impacting the results (Jung, Lyons, Hurwood, & Mather, 2012), and therefore the biological interpretation.…”