genetics presentation - meiping sun and jason dillon - works cited
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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)
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
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
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