Gene Expression Normalization in a Dual-Compartment System: A Real-Time Quantitative Polymerase Chain Reaction Protocol for Symbiotic Anthozoans
(2008)
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Traditional real-time quantitative polymerase chain reaction protocol cannot be used accurately with symbiotic organisms unless the relative contribution of each symbiotic compartment to the total nucleic acid pool is known. A modified ‘universal reference gene’ protocol was created for reef-building corals and sea anemones, anthozoans that harbour endosymbiotic dinoflagellates belonging to the genus Symbiodinium. Gene expression values are first normalized to an RNA spike and then to a symbiont molecular proxy that represents the number of Symbiodinium cells extracted and present in the RNA. The latter is quantified using the number of genome copies of heat shock protein-70 (HSP70) amplified in the realtime quantitative polymerase chain reaction. Gene expression values are then normalized to the total concentration of RNA to account for differences in the amount of live tissue extracted among experimental treatments and replicates. The molecular quantification of symbiont cells and effect of increasing symbiont contributions to the nucleic acid pool on gene expression were tested in vivo using differentially infected sea anemones Aiptasia pulchella. This protocol has broad application to researchers who seek to measure gene expression in mixed organism assemblages.
INTRODUCTION
Coral reefs are ecologically and economically important ecosystems that are threatened by increasing ocean temperatures associated with global climate change (Hoegh-Guldberg 1999; Hoegh-Guldberg et al. 2007). The relationship between anthozoans and endosymbiotic dinoflagellates belonging to the phylogenetically diverse genus Symbiodinium (LaJeunesse 2005; Stat et al. 2006) drives the productivity and structural integrity of the reefs (Muscatine & Porter 1977). Unfortunately, the functionality of coral–dinoflagellate symbioses is particularly sensitive to, and breaks down in the face of, temperature disturbances (Gates 1990), a scenario that manifests as paling of the external colouration of the anthozoan host. This loss of colour can reflect either the loss of dinoflagellate cells and/or a reduction in the amount of chlorophyll per dinoflagellate cell, and is the phenomenon known as bleaching (Brown 1997). Because incidents of mass bleaching are becoming more frequent and predictions for the future temperature environment are bleak, there has been a growing desire to better understand the biological capacity of the anthozoan–dinoflagellate symbiosis to accommodate altered temperature regimes (Hofmann et al. 2005; Edmunds & Gates 2008). Integral to this endeavour is the interest in monitoring expression of genes across a range of temperatures (van Oppen & Gates 2006). However, there is currently no appropriate means of applying real-time quantitative polymerase chain reaction (qPCR) techniques to accurately measure gene expression in dual-compartment (host and symbiont) symbiotic systems (Yellowlees et al. 2008)…
