Aug 10, 2023
Labyrinthulomycete total RNA extraction protocol - hot phenol
- 1Stony Brook University
Protocol Citation: Jackie Collier 2023. Labyrinthulomycete total RNA extraction protocol - hot phenol. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pyo8gwz/v1
Manuscript citation:
Lippmeier, J.C., Crawford, K.S., Owen, C.B., Rivas, A.A., Metz, J.G. and Apt, K.E. (2009), Characterization of Both Polyunsaturated Fatty Acid Biosynthetic Pathways in Schizochytrium sp.. Lipids, 44: 621-630. https://doi.org/10.1007/s11745-009-3311-9
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 10, 2023
Last Modified: August 10, 2023
Protocol Integer ID: 86355
Abstract
Modified from Lippmeier et al. 2009; developed as part of the labyrinthulomycete JGI Community Sequencing Project and Gordon and Betty Moore Foundation Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP)
Safety warnings
Be super careful with hot phenol! It's near the boiling point of phenol, so just a little too hot can be explosive.
Preparing biomass and reagents
Preparing biomass and reagents
Grow up cells, collect, and freeze rapidly - preferably in liquid nitrogen. Store biomass at -80C if not extracting immediately.
This protocol has worked so far for two different thraustochytrids (Aurantiochytrium limacinum ATCC MYA-1381, Schizochytrium aggregatum ATCC 28920) and two Aplanochytrium strains (PBS06 and PBS07).
Prepare extraction buffer
100 mM Tris-HCl pH 7.5
1.5 M NaCl
50 mM Na2-EDTA pH 8.0
20 g per liter CTAB (cetyltrimethylammonium bromide)
8 mg per ml DTT (dithiothreitol) - ADD FRESH JUST BEFORE USE
(note, if you keep these stocks in glass bottles, first soak the bottles with 0.1 M NaOH to inactivate RNAses)
Prepare Tris-equilibrated phenol, pH 4.5-4.8, by warming to 65 C
Prepare acid phenol:chloroform:isoamyl alcohol (pH 4.5-4.8, 125:24:1) by warming to 65 C
Set up to incubate the extraction step at 65 C
Prepare 8M LiCl
Chill 100% ethanol and 70% ethanol
Prepare TE or nuclease-free water
Get a bucket of ice
Extraction steps - REPEAT 3 TIMES (do 3 extractions)
Extraction steps - REPEAT 3 TIMES (do 3 extractions)
Suspend ~1000 micrograms wet weight biomass per ~10 ml extraction buffer by vortexing
(these amounts would yield tens of micrograms of total RNA)
Incubate at 65 C for 5 minutes, mix by vortex or inverting 2 or 3 times
Add equal volume hot (65 C) acid phenol/chloroform/isoamyl alcohol, votes to mix well
Incubate at 65 C for 5 minutes, mix by vortex or inverting 2 or 3 times
Cool the mixture in ice
Centrifuge to separate the phases 4 C, 6,000g, 15 minutes
Move the top phase to a new tube, avoiding the interface
Precipitation steps
Precipitation steps
Add an equal volume of isopropanol and incubate overnight at 4 C
If remaining DNA forms a large fluffy precipitate, remove by spooling onto glass rod
Collect precipitate by centrifugation 4 C, 16,000g, 30 minutes
(may require distributing the sample among several 1.5 ml microfuge tubes and/or multiple spins for each tube to process the volume)
Wash each pellet with 0.5 ml ice-cold 75% ethanol twice, centrifuging 4 C 16,000g, 5 minutes
Allow the pellets to air dry
Dissolve pellets in an appropriate volume, analyze by appropriate method (spectrophotometry, Bioanalyzer); store frozen at -80 C
Optional cleanup steps
Optional cleanup steps
If the RNA contains particulates that do not dissolve, they can be removed by centrifugation 4 C, 6,000g, 5 minutes and transferring the supernatant to a fresh tube.
Even if the RNA does not have obvious DNA, a DNAse treatment step is nonetheless recommended
If the RNA contains large amounts of DNA or other contaminants, it can be removed by selectively precipitating the RNA with 17 microliters of 8M LiCl per 50 microliters RNA
Incubate -20 C 60 minutes
Collect RNA by centrifugation 4 C, 16,000g, 20 minutes
Wash each pellet with 0.5 ml ice-cold 75% ethanol twice, centrifuging 4 C 16,000g, 5 minutes
Allow the pellets to air dry
Dissolve pellets in an appropriate volume, analyze by appropriate method (spectrophotometry, Bioanalyzer); store frozen at -80 C