Apr 28, 2020
RNA Extraction Protocol for Aurantiochytrium limacinum
- 1State University of New York at Stony Brook;
- 2Stony B
- Collier Lab
Protocol Citation: Michael Horowitz, Mariana Rius, Jackie Collier 2020. RNA Extraction Protocol for Aurantiochytrium limacinum. protocols.io https://dx.doi.org/10.17504/protocols.io.bffgjjjw
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: April 22, 2020
Last Modified: April 28, 2020
Protocol Integer ID: 36040
Materials
MATERIALS
Liquid Nitrogen
TRIZOL reagent
Chloroform
Monarch Total RNA Miniprep KitNew England BiolabsCatalog #T2010S
Ethanol
Safety warnings
Take safety precautions when using liquid nitrogen. Make sure to conduct Trizol and chloroform steps in fume hood.
Prepare cells
Prepare cells
Start a preculture 4 days prior to extraction by inoculating 5 ml of 790 with a colony of Aurantiochytrium limacinum (ATCC MYA-1381). Incubate overnight at 28 C 171 rpm.
Use preculture to inoculate 20 ml of 790 in a 250 ml flask. Culture for three days at 28 C, 171 rpm.
Determine volume of culture needed to reach 5*10^7 cells via cell count.
Place volume needed into a microcentrifuge tube and centrifuge down at 13000rpm for two minutes at room temperature. Discard supernatant.
Place pellet in liquid nitrogen for 3-5 minutes.
Trizol Extraction
Trizol Extraction
Resuspend pellet in 750ul Trizol lysis buffer and incubate on ice for 1 hour and 30 minutes.
Add 150ul chloroform in fume hood to the pellet and let sit for 2 minutes.
Vigorously shake the mixture and centrifuge at 13000rpm for 5 minutes at 4C.
Pipette upper aqueous layer to a sterile microcentrifuge tube on ice.
Add an equal volume of >95% ethanol to the upper aqueous layer and mix well.
Monarch Kit RNA Extraction
Monarch Kit RNA Extraction
Use NEB Monarch Total RNA Miniprep Kit from here. All centrifugation steps should be carried out at 16,000rcf.
Take an RNA purification column and collection tube. Place the mixture (the upper aqueous layer with ethanol) from the microcentrifuge tube to the purification column.
Centrifuge for 30 seconds and discard flow-through.
Add 500ul RNA Wash Buffer and spin for 30 seconds. Discard flow-through.
In a separate RNase-free microcentrifuge tube, combine 5ul DNase 1 provided in the Monarch Kit with 75ul DNase 1 Reaction Buffer. Pipette the mixture directly onto the column matrix.
Incubate for 15 minutes at room temperature.
Add 500ul RNA Priming Buffer and spin for 30 seconds. Discard the flow-through.
Add 500ul RNA Wash Buffer and spin for 30 seconds. Discard the flow-through.
Add another 500ul RNA Wash Buffer and spin for two minutes. Transfer the column to a new RNase-free microcentrifuge tube.
Add 30ul of Nuclease-free Water directly to the column matrix and spin for 30 seconds.
Pipette the flow-through back into the column matrix and spin again for 30 seconds.
Remove column matrix, close the microcentrifuge tube, and label it. Store the eluted RNA at -20C for short-term storage or at -80C for long-term storage.
RNA yields for GPY grown cells are usually around a 1000ng/ul and for 790-grown cells are much lower around 50-100ng/ul. Lower yield for 790 cells are possibly due to excessive cell clumping.