Multi-macromolecular Extraction from Endosymbiotic Anthozoans

Abstract

Obligately symbiotic associations between reef-building corals (anthozoan cnidarians) and photosynthetically active dinoflagellates of the family Symbiodiniaceae comprise the functional basis of all coral reef ecosystems. Given the existential threats of global climate change toward these thermo-sensitive entities, there is an urgent need to better understand the physiological implications of changes in the abiotic milieu of scleractinian corals and their mutualistic algal endosymbionts. Although initially slow to leverage the immense breakthroughs in molecular biotechnology that have benefited humankind, coral biologists are making up for lost time in exploiting an array of ever-advancing molecular tools for answering key questions pertaining to the survival of corals in an ever-changing world. In order to comprehensively characterize the multi-omic landscape of the coral holobiont—the cnidarian host, its intracellular dinoflagellates, and a plethora of other microbial constituents—I introduce a series of protocols herein that yield large quantities of high-quality RNA, DNA, protein, lipids, and polar metabolites from a diverse array of reef corals and endosymbiotic sea anemones. Although numerous published articles in the invertebrate zoology field feature protocols that lead to sufficiently high yield of intact host coral macromolecules, through using the approach outlined herein one may simultaneously acquire a rich, multi-compartmental biochemical pool that truly reflects the complex and dynamic nature of these animal–plant chimeras.

Appendices

Appendices

Appendix Sheet 1

Printable protocols for RNA, DNA, and protein extractions. Note that the blank spaces are to be used to either place check marks (to demonstrate completion of the respective step) or to fill in pertinent details, namely with respect to temperatures (temp.), times, and volumes. Alternative RNA and DNA spin column kits can be substituted.

RNA Extraction             Date: _________

• Homogenization and Phase Separation

  • 1. Homogenized in: LN₂_____ TRIzol®_____ TRI-Reagent®______ Other______

    • (a) w/: mortar and pestle___ micro-pestle___ tissue lyser__ bead mill__ other___

  • 2. Incubated at _______(temp.) for _______(time) after vigorous vortexing.

    • (a) w/: shaker table_____ tissue lyser_____

  • 3. Added 200 μL of chloroform and incubated at RT for _______(time). w/: new tube_______

  • 4. Spun at 12,000 × g for 15 min at 4  °C and transferred aqueous phase to new tube______.

• Precipitation

  • 5. Precipitated w/ 250 μL of isopropanol and 250 μL of HSS at ____(temp.) for ______(time) and spun at 12,000 × g for 10 min at 4  °C.

    • (a) w/: Pellet Paint™ _____

• Purification

  • 6. Resuspended pellet in ____ μL of lysis buffer A and ____ μL of 100% ethanol.

  • 7. Added ____ μL to _________(manufacturer’s name) RNA kit spin column.

  • 8. Followed manufacturer’s recommendations and incubated columns in 60 °C oven.

    • (a) On-column DNase digestion______(yes or no)

  • 9. Eluted into _____ μL of DEPC-treated water.

DNA Extraction

• Phase Separation

  • 1. Added 500 μL of BEB and incubated on shaker table for _______(time).

  • 2. Spun at 12,000 × g for 10 min at 4 °C and transferred aqueous phase to new tube______.

• Precipitation

  • 3. Precipitated DNA w/ 60 μL of 3 M NA acetate and 600 μL of isopropanol at _____(temp.) for ____(time).

    • (a) w/: 2 μL of Pellet Paint™_____(yes or no).

  • 4. Spun at 12,000 × g for 10 min at 4 °C_____.

• Purification

  • 5. Resuspended pellet in a minimum volume of 100 μL of the first buffer of the preferred DNA clean-up kit (e.g., PCR-A buffer from Axygen’s PCR clean-up kit)_____.

  • 6. Carried out spins and washes as recommended by manufacturer_____.

  • 7. After evaporating residual ethanol in 60 °C oven, eluted DNA into ___μL of pre-warmed (to 60 °C) “eluent” (must include Tris at a pH of at least 8 for best DNA elution).

Protein Extraction

• 8. Precipitated protein in 1.5 mL of acetone at ______(temp.) for _______(time).

• 9. Spun at 12,000 × g for 10 min at 4 °C____.

• 10. Washed pellet 3× w/ PWI and 1× w/ PWII at 8000 × g for 5 min at 4 °C, and dried on benchtop for _____ min.

• 11. Resuspended protein in _____μL of______ buffer, sonicated for _____ min, boiled for ____ min at 100 °C, and transferred _____μL to each of ____tubes.

• 12. Quantified ____μL with the 2-D Quant kit, ____ μL with a Bradford assay, ___ μL with a BCA assay, or ___ μL with the Qubit protein assay kit.

Appendix Sheet 2

Printable sheets for RNA, DNA, and protein quantification and quality control analyses. A spectrophotometer or, preferably, a bioanalyzer can be used to generate these (or comparable) data. Subscripts correspond to the technical replicate number. BSA = bovine serum albumin (the most common standard used in protein quantification assays). QC = quality control.