Gene expression normalization in a dual-compartment system: a real-time quantitative polymerase chain reaction...

Area of Science:

• Marine Biology
• Molecular Biology
• Genomics

Background:

• Symbiotic organisms, like corals, present challenges for gene expression studies due to the contribution of multiple genomes.
• Accurate quantification of gene expression in host-symbiont systems requires normalization to account for the relative abundance of each partner.

Purpose of the Study:

• To develop and validate a modified 'universal reference gene' protocol for accurate gene expression analysis in anthozoans harboring Symbiodinium.
• To establish a method that normalizes gene expression data to both RNA concentration and a symbiont molecular proxy.

Main Methods:

• Developed a protocol normalizing gene expression values first to an RNA spike and then to a symbiont molecular proxy.
• Quantified Symbiodinium cells using genome copy number of heat shock protein-70 (HSP70) via real-time quantitative polymerase chain reaction.
• Normalized gene expression to total RNA concentration to control for variations in extracted live tissue.

Main Results:

• Successfully tested the protocol in vivo using differentially infected sea anemones (Aiptasia pulchella).
• Demonstrated the ability to molecularly quantify symbiont cells and assess the impact of symbiont load on gene expression.
• Validated the protocol's effectiveness in mixed organism assemblages.

Conclusions:

• The modified protocol provides accurate gene expression measurements in corals and sea anemones by accounting for endosymbiotic dinoflagellates.
• This method is broadly applicable to researchers studying gene expression in any mixed organism system.
• Improved accuracy in gene expression analysis is crucial for understanding host-symbiont interactions.