Gene expression normalization in a dual-compartment system: a real-time quantitative polymerase chain reaction protocol for symbiotic anthozoans

Gene expression normalization in a dual-compartment system: a real-time quantitative polymerase chain reaction protocol for symbiotic anthozoans

Year: 

2009
Authors: 
Mayfield, A. B.Hirst, M. B.Gates, R. D.

Source: 

Molecular Ecology Resources

Abstract: 

Traditional real-time quantitative polymerase chain reaction protocols 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 real-time 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.

Volume: 

9

Issue: 

2

Pages: 

462-470

ISBN: 

1755-098X

Publication Type: 

Journal Article

ISI Number: 

ISI:000263333100003

Research Areas: