Abstract
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to confirm 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 differentially expressed at different ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profiles of nine candidate reference genes from the brackish water flea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four different 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 significantly different pattern in suite of candidate reference genes. This affected the results of genes EcRA and GST, which are involved in development and detoxification mechanisms, respectively. Our finding is the first step towards establishing a standardized real-time qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.
Highlights
Environmental pollutants, such as heavy metals and persistent organic pollutants, have been of great concern because of their persistence in aquatic environments and harmful effects on aquatic o rganisms[1,2]
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), EF-α, and 18S rRNA, the most popular genes still preferred in aquatic invertebrate for normalization, remain u nvalidated[15], and some genes suggested as reference genes can be modulated by different environmental s tresses[16], indicating that even housekeeping genes may be involved in pathways other than cellular h omeostasis[17]
We investigated the stability of the nine candidate reference genes under different ages (24-h, 4-day, 7-day, and 10-day) and measured the relative expression of target genes based on the conditions of reference genes
Summary
Environmental pollutants, such as heavy metals and persistent organic pollutants, have been of great concern because of their persistence in aquatic environments and harmful effects on aquatic o rganisms[1,2]. Brackish water species have a wide range of adaptations to environmental physico-chemical conditions, such as salinity and temperature, and are considered suitable for monitoring and risk assessment of various pollutants originating from land and marine environments. The brackish water flea Diaphanosoma celebensis, that belongs to Crustacea, Branchiopoda, and Cladocera, is one of the species of interest to many scientists as potential non-model species for marine p ollution[22,23,24] They are widely distributed in tropical Asia at a wide range of salinity and temperature. We selected nine common housekeeping genes as candidate reference genes from previous studies[27,28,29,30] and compared their stability in expressions to identify appropriate reference genes under conditions such as chemical exposure and age. We investigated the stability of the nine candidate reference genes under different ages (24-h, 4-day, 7-day, and 10-day) and measured the relative expression of target genes based on the conditions of reference genes
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