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 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.

Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi

“…In the last decades, the genes known as housekeeping genes were broadly used as internal controls for the normalization of RNA levels for Northern blotting, RNAse protection and RT-qPCR analyses, because it was assumed that these genes are constitutively expressed and regulated, but no studies indeed evaluated the stability of expression of these genes under various experimental conditions 7,14 . Moreover, it has been shown that the expression levels of these housekeeping genes vary depending on the conditions and tissues in which are expressed [15][16][17][18][19] . Therefore, it is hard to select a perfect RG that would be consistently expressed in all tissues and cell types without being in uenced by internal or external factors.…”

Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi

“…In the last decades, genes known as housekeeping genes were broadly used as internal controls for the normalization of RNA levels for Northern blotting, RNAse protection, and RT-qPCR analyses because it was assumed these genes are constitutively expressed and regulated, but no studies have evaluated the stability of expression of these genes under various experimental conditions 11 , 23 . Moreover, the expression levels of these housekeeping genes vary depending on the conditions and tissues expressed 24 – 28 . Therefore, it is difficult to select a perfect RG that would be consistently expressed in all tissues and cell types without being influenced by internal or external factors.…”