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Total RNA Extraction from Dinoflagellate Symbiodinium Cells
Published: Jun 5, 2018 DOI: 10.21769/BioProtoc.2866 Views: 4535
Edited by: Adam Idoine Reviewed by: Manuel Aranda
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Abstract
Dinoflagellates are unicellular algae that can have photosynthetic or nonphotosynthetic lifestyles. Dinoflagellates in the genus Symbiodinium can enter endosymbiotic associations with corals, providing the metabolic basis for the highly productive and biologically diverse coral-reef ecosystems (Hoegh-Guldberg, 1999), as well as with other cnidarians, including sea anemones and jellyfish, and non-cnidarian hosts (Trench, 1993; Lobban et al., 2002; Mordret et al., 2016).
Here, I describe a protocol for isolating total RNA from Symbiodinium cells.
Coral
Symbiosis
RNA extraction
DNA
Materials and Reagents
- Gloves (E&K Scientific Products, catalog number: EK400-S )
- Pipette tips 10/20 μl (USA Scientific, catalog number: 1120-3810 ), 200 μl (USA Scientific, catalog number: 1120-8810 ), 1,000 μl (USA Scientific, catalog number: 1126-7810 )
- Microcentrifuge tube 1.5 ml (E&K Scientific Products, catalog number: 280150 )
- Microcentrifuge tube 2.0 ml (Thermo Fisher Scientific, catalog number: 3463 )
- Symbiodinium SSB01
- Diethylpyrocarbonate (DEPC) treated water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9922 )
- Liquid nitrogen
- Glass beads, acid-washed (Sigma-Aldrich, catalog number: G8772 )
- Chloroform:isoamyl alcohol (24:1, v/v, Sigma-Aldrich, catalog number: C0549 )
- Phenol:chloroform:isoamyl alcohol (25:24:1, v/v, Sigma-Aldrich, catalog number: P2069 )
- Ethanol (Absolute, PHARMCO-AAPER, catalog number: 111000200 )
- 8 M LiCl (Sigma-Aldrich, catalog number: L7026 )
- RNase-Free DNase (QIAGEN, catalog number: 79254 )
- Agarose (Thermo Fisher Scientific, Thermo Scientific TM, catalog number: 17850 )
- Phenol solution (Sigma-Aldrich, catalog number: P4682 )
- 3 M sodium acetate (VWR, catalog number: 97062-812 )
- Coral Pro Salt (Red Sea)
- Tris 1 M solution, pH 9.0 (VWR, catalog number: 97062-940 )
- Sodium chloride, NaCl (Sigma-Aldrich, catalog number: S9888 )
- Tris (hydroxymethyl) aminomethane (Sigma-Aldrich, catalog number: 252859 )
- Ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, catalog number: E9884 )
- Sodium dodecyl sulfate, SDS (Sigma-Aldrich, catalog number: L3771 )
- Artificial Sea Water (see Recipes)
- RNA Lysis buffer (pH 9.0) (see Recipes)
- RNA lysis buffer (pH 7.0) (see Recipes)
Equipment
- Glass Erlenmeyer flask
- RNase-Free glass bottle
- Pipettes
0.2-2 μl (VWR, catalog number: 89079-960 )
2-20 μl (VWR, catalog number: 89079-964 )
20-200 μl (VWR, catalog number: 89079-970 )
100-1,000 μl (VWR, catalog number: 89079-974 )
5 ml (E&K Scientific Products, catalog number: EK-67044 ) - Vortex (VWR, catalog number: 97043-562 )
- Incubator
- -20 °C freezer
- Mini bead beater (Cole-Parmer, catalog number: EW-36270-07 )
- Oven
- Centrifuge (Sorvall, model: Sorvall RC 5C )
- Electrophoresis instrument
Procedure
Category
Microbiology > Microbe-host interactions > Algae
Molecular Biology > RNA > RNA extraction
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