Abstract
BackgroundGenomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation.ResultsEight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable.ConclusionWe present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements.
Highlights
Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology
Quantitative Reverse Transcriptase PCR (QRT-PCR) is a valuable tool for ecological studies as it provides a relatively straightforward way to measure the direct transcriptional response of an organism exposed to different treatments [2]
An example of the different expression levels in treatments and conditions is given in Figure 1 for ACTb and the differential metallothionein gene (MT) of O. cincta
Summary
Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. Quantitative Reverse-Transcriptase Polymerase Chain Reaction (QRT-PCR) is a technique to estimate gene expression levels. This technique is often used to confirm high throughput systems like microarrays. Its application has mainly been limited to small numbers of genes per experiment due to constraints of low throughput coinciding with relatively high costs per assay This is about to change, as high throughput QRT-PCR systems using small volume (capillary) PCR are becoming available [1]. QRT-PCR is a valuable tool for ecological studies as it provides a relatively straightforward way to measure the direct transcriptional response of an organism exposed to different treatments [2]. Roelofs et al [7] conducted a QRT-PCR study to assess the relevance of transcriptional regulation in the adaptive evolution of stress tolerance
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
