Gene Expression Normalization in a Dual-compartment System: a Real-time Quantitative Polymerase Chain

Reaction Protocol For Symbiotic Anthozoans

Meiping Sun and Jason Dillon

Introduction Coral reefs are ecologically and economically important

ecosystems

“Bleaching” of Coral reefs

Biological capacity of the anthozoan-dinoflagellate symbiosis to accommodate altered temperature regimes

Monitoring expression of genes across a range of temperatures

Goal—Developing the capacity to quantify the endosymbiont contribution to a given extraction

Challenges ◦Common real-time quantitative

polymerase chain reaction (qPCR) technique pitfalls

◦Varying RNA composition of the DNA/RNA samples

“Symbiont molecular proxy (SMP)”

RNA/DNA spiking approach◦Control for other technical issues

Total RNA and total protein (evaluate quantity of biological material)

Proteins, DNA, and RNA

Materials and methodsCollections of anemones and corals

and isolation of dinoflagellate symbionts◦Coconut Island◦Dinoflagellate cells were pelleted by

centrifugation◦Dinoflagellate cell pellets were re-

suspended in FSW◦Haemacytometer counts

Aiptasia pulchella infection◦A. pulchella were artifically infected

with Symbiodinium For each of five clonal lines, 6

anemones were infected, 2 uninfected as controls

…….

Results – HSP70 CorrelationsM. capitata

◦<0.5% dinoflagellate cells w/ membrane damage

◦r2>0.87 (HSP70 genome copies vs. symbiont cells)

◦r2=0.97 (coral-derived dilution series, 0.125–1 × 106 cells)

◦2-2.5 x 106 SMP is too highr2=0.88 (A. pulchella - 0.125 – 2 × 106 cells)

Figure 2 - Freshly isolated Symbiodinium cell dilutions and HSP70 genome copies

HSP70 Levels After InfectionAfter infection HSP70 increases,

regardless of:◦Non-normalization◦Exogenous DNA spike only◦DNA spike & protein◦DNA spike & total RNA

Uninfected controls remained uninfected

Figure 3 - Symbiodinium densities and HSP70 genome copies in Aiptasia pulchella

Figure 4 - Symbiodinium HSP70 expression in Aiptasia pulchella

Data Relevance

rt-PCR (qPCR) has been used to identify adaptive advantages in symbiotes

Bleached anthozoans account for most RNA due to up-regulation

Must interpret data in vivoStrong correlation for anemone-derived

symbionts (r2=0.92) & coral-derived symbionts (r2=0.97)◦Valid for 106 cells

Discussion – Best Protocol

Standard errors for 2 and 2.5 × 106 cell counts◦Too great for predictive abilities◦Pipetting errors

Findings demonstrate need to normalize gene expression to SMP◦ If not, no accurate measurement of symbiotic

ratioNormalization to RNA better than to

protein◦Pipetting errors

Discussion - HousekeepingHousekeeping genes (cell scaffolding

and volume)◦ACTB & a-tubulin◦Thermal stress – cell volume changes◦Breakdown of symbiosis

“Heat Specific Protein (HSP) 70”

Efficiency of StudyNormalizing to DNA/RNA spikes

◦Extraction/reverse transcription efficiencySMP account for differential extraction

of SymbiodiniumTotal RNA proxy for comparison

ConclusionAnthozoans and dinoflagellates are in

symbiosisSymbiodinium undergoes

photosynthesis, while anthozoans undergo respiration

Heat stress causes production of HSP70 in Symbiodinium

Conclusion contd.

qPCR quantification can be carried out in a “dual-compartment’ system

Must be quantified and normalizedLinear correlation up to 106 cells

Further Questions/EndeavorsHow can one quantify symbiont levels

that exceed 106 cells?Protocol now developed for future

researchFind possible alternative symbiotic

relationshipsDetermine levels of heat stress in

such organisms

Works Cited

Mayfield, Anderson, Marissa Hurst, and Ruth Gates. Gene expression normalization in a dual-compartment system: a real-time quantitative polymerase chain reaction protocol for symbiotic anthozoans. Hoboken, NJ: Blackwell Publishing, 2009.