Validation of suitable reference genes for quantitative real-time PCR normalization in Crassostrea gigas spat stage during toxic dinoflagellates exposure//Validación de genes de referencia adecuados para la normalización de PCR cuantitativa en tiempo real de juveniles de Crassostrea gigas expuestos a dinoflagelados tóxicos
DOI:
https://doi.org/10.18633/biotecnia.v22i2.1250Keywords:
Gene expression, reference gene, normalization, toxic dinoflagellates, Crassostrea gigasAbstract
The quantitative real-time polymerase chain reaction is a widely used method for gene expression analysis requiring carefully selected reference genes to ensure data validity. Regardless of several studies on gene expression bivalves and particularly Crassostrea gigas, has not been fully investigated regarding the evaluation of reference genes suitable for normalization of expression analysis. In this study, five candidate reference genes: actin, β tubulin, α subunit of elongation factor 1, glyceraldehyde-3-phosphate dehydrogenase and 28S ribosomal RNA were analyzed, to determine the most suitable reference genes, after of Crassostrea gigas spat were fed with Gymnodinium catenatum and Prorocentrum lima in mixed and compared to non-toxic diet Isochrysis galbana. The results showed that β-tub and ef-1α were the most stable genes for oysters feed with a mixed diet of P. lima and I. galbana. The gapdh and 28S rRNA were the most stable genes for oysters feed with G. catenatum and I. galbana. In addition, the selection of optimal reference genes during dinoflagellates exposure was verified by analyzing the expression level of trypsin and cytochrome c oxidase I target genes. Our study could be beneficial for future studies on gene expression in C. gigas.
RESUMEN
La reacción en cadena de la polimerasa cuantitativa en tiempo real es un método ampliamente utilizado para el análisis de expresión génica, requiere genes de referencia cuidadosamente seleccionados para garantizar la validez de los datos. A pesar de los estudios sobre expresión génica en bivalvos y particularmente en Crassostrea gigas, existe escasa información sobre la evaluación de los genes de referencia adecuados para la normalización del análisis de expresión. En este estudio se analizaron cinco genes candidatos de referencia: actina, β tubulina, subunidad α del factor de elongación 1, gliceraldehído-3-fosfato deshidrogenasa y ARN ribosomal 28S, para determinar los genes más adecuados, en juveniles
de Crassostrea gigas alimentado con una dieta mixta de Gymnodinium catenatum y Prorocentrum lima en comparación con una dieta no tóxica de Isochrysis galbana. Los resultados mostraron que β tub y ef1 α fueron los genes más estables para C. gigas alimentado con una dieta mixta de I. galbana y P. lima; los genes más estables para los ostiones alimentados con I. galbana y G. catenatum fueron gapdh y 28S rRNA. Además, la selección de los genes de referencia durante la exposición a dinoflagelados tóxicos se verificó analizando el nivel de expresión de los genes blanco tripsina y citocromo c oxidasa I. El presente estudio será de gran utilidad para futuros estudios sobre análisis de expresión génica en juveniles de C. gigas.
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