Validation of reference genes for quantitative real-time PCR in chemical exposed and at different age’s brackish water flea Diaphanosoma celebensis

Abstract

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to confrm relative gene expression levels by comparison, and rule out variations that might occur in analytical procedures. The best option is to use a reference gene whose expression level is stable across various experimental conditions to compare the mRNA levels of a target gene. However, there is limited information on how the reference gene is diferentially expressed at diferent ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profles of nine candidate reference genes from the brackish water fea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four diferent algorithms showed similar stabilities of genes for chemical exposures in the case of limited conditions using the same developmental stage (H2A was stable, whereas Act was fairly unstable in adults), while the results according to age showed a signifcantly diferent pattern in suite of candidate reference genes. This afected the results of genes EcRA and GST, which are involved in development and detoxifcation mechanisms, respectively. Our fnding is the frst step towards establishing a standardized realtime qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.