Nov 21, 2020
Coral tissue and skeleton Trizol RNA extraction
- 1Marine Biological Laboratory
Protocol Citation: Molly A Moynihan 2020. Coral tissue and skeleton Trizol RNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bi9ikh4e
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 02, 2020
Last Modified: November 21, 2020
Protocol Integer ID: 39946
Abstract
This protocol was modified from the TRIzol protocol provided by Invitrogen.
Materials
MATERIALS
Ethanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500
nuclease free water
RNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
5M NaClAmbionCatalog #AM9760G
Sodium CitrateSigmaCatalog #71402
RNaseZap™ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
TRIzol™ ReagentThermo FisherCatalog #15596026
Phasemaker™ TubesThermo FisherCatalog #A33248
GlycoBlue™ Coprecipitant (15 mg/mL)Thermo FisherCatalog #AM9516
Isopropanol Molecular Biology Grade
Chloroform Molecular biology grade
Refrigerated centrifuge for 2mL tubes
Solutions to prepare ahead of time
Solutions to prepare ahead of time
Make high-salt buffer (0.8M NaCitrate/1.2 M NaCl).
Add 23.5 g NaCitrate to 24 mL of 5 Molarity (M) NaCl .
Add water to a final volume of 100 mL .
Autoclave and aliquot into working solutions. Each sample will need 250 µL high salt buffer .
Set Up
Set Up
Cleaning and tips to avoid contamination:
Clean UV hood and fume hood with bleach, ethanol, RNaseZap, and sterilize UV hood for 15 minutes.
Clean all pipette tips with bleach, ethanol, RNaseZap.
Keep UV hood and fume hood pipettes separate.
Use reagents that are dedicated for RNA work.
Prepare solutions and aliquots in clean, sterilized UV hood.
Use only nuclease free plasticware.
Use filter pipette tips designated exclusively for RNA work.
Warning:
Trizol reagent contains guanidinium thiocyanate and acid phenol. All procedures involving the transfer of Trizol or chloroform should be performed in a chemical fume hood. Waste should be treated as hazardous and disposed of properly.
Using molecular biology grade ethanol, prepare fresh 75 % (v/v) ethanol (2mL per sample). Place 75% ethanol in -20 °C freezer so that it will be cold when used in Step 18.
Cool down centrifuge to 4 °C .
RNA/DNA Separation
RNA/DNA Separation
Thaw samples.
Since coral tissue was previously homogenized with the tissue tearor during separation, it should not be too viscous when pipetted. It might be thick, but you should be able to pipette it with ease. Homogenize again with tissue tearor if it is too viscous. When the tissue is not well homogenized, it will be difficult to separate the aqueous phase in Step 10.
In fume hood, add 1 mL Trizol to sample aliquots prepared during tissue/skeleton separation (approximately 200-500μl of tissue solution and 200-500cm3 of skeleton).
If aliquots have more sample than desired for extraction, transfer sample into a new nuclease free 2mL tube and add 1mL Trizol.
If desired, Phasemaker tubes can be used here instead of 2mL microcentrifuge tubes to facilitate phase separation and help prevent DNA contamination.
For skeletal samples, add 1 µL to 5 µL of 6M HCl (18.5% HCl) to sample/Trizol mixture.
Vortex sample/Trizol mixture for 00:00:30 . It is important thoroughly homogenize the sample in Trizol. Incubate samples for 00:05:00 to allow for complete dissociation of the nucleoproteins complex.
In fume hood, add 200 µL chloroform per 1mL of Trizol reagent. Shake samples vigorously by hand and then vortex briefly. The solution should be well mixed and milky.
Incubate for 00:03:00 at room temperature.
Centrifuge 12000 x g, 4°C, 00:15:00 . The mixture will separate into a lower red phenol-chloroform, an interphase, and a colorless upper aqueous phase.
In fume hood, carefully transfer the upper aqueous phase containing the RNA to a new 2mL DNase/RNase free tube. Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase.
RNA Isolation
RNA Isolation
In fume hood, add 250 µL isopropanol and 250 µL high salt solution (0.8 NaCitrate/1.2M NaCl) to the aqueous phase, per 1mL of Trizol used in lysis.
Adding the high salt solution allows polysaccharides and proteoglycans to remain in soluble form (Chomczynski & Mackey, 1995), preventing them from co-precipitating with the pellet.
Add 1 µL glycoblue (20mg/ul) . Vortex briefly.
Incubate at room temperature for 00:10:00 .
Centrifuge at 12000 x g, 4°C, 00:10:00 . The RNA precipitate forms a blue gel-like pellet on the side or bottom of the tube. If glycoblue was not used, the pellet will be clear/white.
Without disturbing the pellet, remove and discard the supernatant. The pellet might be barely visible so use caution when pipetting. It is helpful to know the side of the tube your pellet will be on, based on its position in the centrifuge.
(After this step, move to the UV sterilized hood, instead of the fume hood).
Using the ethanol prepared in Step 3, resuspend the pellet in 1 mL cold 75% ethanol per 1mL of Trizol. Vortex briefly.
Centrifuge at 7500 x g, 4°C, 00:05:00 .
Without disturbing the pellet, remove and discard the supernatant.
Repeat the ethanol wash by resuspend the pellet in 1 mL cold 75% ethanol per 1mL of Trizol. Vortex briefly.
Centrifuge at 7500 x g, 4°C, 00:05:00 .
Without disturbing the pellet, remove and discard the supernatant.
Air dry pellet for 00:05:00 to 00:10:00 by leaving the lid open.
Solubilize RNA by resuspending the pellet in 50 µL nuclease free water by pipetting up and down. Incubate on heat block at 55 °C for 00:10:00
Note: For some coral species, a white insoluble pellet may form during precipiation. Using a clean and concentrator kit (e.g. Zymo) can help to remove insoluable co-precipitants and improve RNA solubilization. See optional section below.
Check RNA yields.
RNA concentration should range from 50-300 ng/μl. If yield is low (<50 ng/μl) use clean and concentrate protocol below.
Store at -80 °C .
RNA Clean & Concentrate
RNA Clean & Concentrate
For 50μl of sample, add 100 µL RNA Binding Buffer and vortex briefly.
Add 150 µL 100% Ethanol and vortex briefly.
Transfer solution to spin column and collection tube.
Centrifuge at 10000 x g, Room temperature, 00:00:30 and discard flow-through.
Add 400 µL RNA Prep Buffer to column and centrifuge at 10000 x g, Room temperature, 00:00:30 . Discard the flow-through
Add 700 µL RNA Wash Buffer to column and centrifuge at 10000 x g, Room temperature, 00:00:30 . Discard the flow-through
Centrifuge at 10000 x g, Room temperature, 00:01:00 to fully remove wash buffer.
Transfer the column into a new nuclease-free tube.
Add desired volume of nuclease free water to column and incubate at room temperature for 5 minutes.
Note: For reverse transcription, aim to have ≥ 500ng of RNA for the RT reaction and ≥ 500ng of RNA for the noRT control. For solutions with low yield, eluding in 35 µL nuclease free water it recommended, so that each RT/noRT reaction can contain 12μl of sample, leaving 11μl that can be used for quantification and to check for DNA contamination.
Centrifuge at 10000 x g, Room temperature, 00:00:30 .