Aug 20, 2019
RNA Isolation from Plant Tissue Protocol 14: Ambion Trizol RNA Extraction in Microcentrifuge Tubes with Turbo DNAfree Digestion
Peer-reviewed method
- GigaScience Press
External link: https://doi.org/10.1093/gigascience/giz126
Protocol Citation: Eric Carpenter: RNA Isolation from Plant Tissue Protocol 14: Ambion Trizol RNA Extraction in Microcentrifuge Tubes with Turbo DNAfree Digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.4u6gwze
Manuscript citation:
Carpenter EJ, Matasci N, Ayyampalayam S, Wu S, Sun J, Yu J, Jimenez Vieira FR, Bowler C, Dorrell RG, Gitzendanner MA, Li L, Du W, K Ullrich K, Wickett NJ, Barkmann TJ, Barker MS, Leebens-Mack JH, Wong GK. Access to RNA-sequencing data from 1,173 plant species: The 1000 Plant transcriptomes initiative (1KP). Gigascience. 2019 Oct 1;8(10):giz126. doi: 10.1093/gigascience/giz126.
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
These protocols were used for RNA extraction from plant tissues in order to support the One Thousand Plants initiative's work to produce RNA-Seq transcriptomes from a diverse collection of plant samples.
Created: June 27, 2019
Last Modified: August 22, 2019
Protocol Integer ID: 25214
Keywords: RNA, RNA extraction, RNA isolation, plant tissue, woody plants, aquatic plants
Abstract
Implemented by: Ingrid Jordon-Thaden and Nicholas Miles (Soltis Labs)
Note
Concise version of Protocol (longer version available on request from Ingrid Jordon-Thaden, ingridejt@gmail.com)
This procedure eliminates the mortar and pestle homogenization of tissues and instead grinds tissue in 2 ml microcentrifuge tubes. The method closely follows Ambion’s protocols and could be used in a 96-well format. This method worked great for species that proved to be difficult to extract with other methods (i.e. woody and aquatic plants).
Note
Both the addition of β-mercaptoethanol in extraction and high salts (recommended by Ambion: 0.8 M sodium citrate and 1.2 M NaCl) in precipitation were tried with this method and yield or quality was not affected. The addition of Sarkosyl significantly improved both yield and quality.
This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6)
Attachments
Materials
MATERIALS
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
TURBO DNA-free™ KitThermo Fisher ScientificCatalog #AM1907
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
Listed below are two slightly different methods for tissue collection: one directly in microcentrifuge tubes, and one in 50 ml tubes.
Leaf Collection in microcentrifuge tubes (best to use 60 to 100 mg of tissue for high throughput)
Pre-label RNAase free 2 mL tubes, place 5 zirconia beads (pre-baked at 200 °C for 04:00:00 ) in each tube and place in boxes. Cut leaf tissue and put into the tube, then directly into small cooler with liquid N2. Store the boxes with the leaves in -80 °C freezer.
Leaf collection in 50 mL Falcon tubes (for large collections so many extractions can be made on the same sample if necessary)
Identify the species to collect, give it a collection number, and write the number of the 50 ml Falcon tube. Use a scissor or pin to cut a hole in the top of the tube. Fill the tube with N2 and place in cooler with N2 and rack. Clean scissors with Ethanol and RNAzap. Cut the youngest leaf tissue and immediately put into the Falcon tube for freezing. Take a
specimen of the plant for a voucher. Place the tubes in the -80 °C freezer.
Note
All centrifuge steps at 4 °C unless otherwise specified.
Clean all surfaces and equipment with 20 % bleach, 95 % ethanol, and RNase Zap, and place supplies in hood.
Prepare sample tubes with labels.
Fill microcentrifuge tubes with 5 zirconia beads (if they have not been done before tissue collection).
Prepare aliquots of the following solutions into 50mL Falcon tubes: 75 % ethanol, 100 % chloroform, 100 % isopropanol.
Fill dewar with liquid N2.
Get samples from -80 °C and place them in the black shaker block that is sitting in a bath of liquid N2 (if already in microcentrifuge tubes), and check labels.
Prepare frozen tissue in microcentrifuge tubes from 50 ml Falcons (if tissue is not in them already).
Fill two small coolers with liquid nitrogen (one for microcentrifuge tubes, the other for Falcon tubes).
Doing about 3 or 4 plants at a time, use forceps or a spatula to move 60 mg to 100 mg of tissue into the microcentrifuge tubes from the 50 ml Falcon tubes.
Get a weight for each of the samples, add more if necessary.
This kit requires 60 mg to 100 mg of plant tissue.
Note
If you are doing aquatic plants use 2x the weight since so much of the weight is water.
Repeat these steps until you have finished all of the samples.
Before pulverizing the frozen tissue, check each tube for beads, making sure they are easily moving within the tube.
Taking the black shaker block with the 24 samples, place tightly into the automatic shaker, doing this quickly as to not allow thawing.
Shake for 00:02:00 (they will stay frozen).
Place block back into the liquid nitrogen cooler if needed to shake another 00:02:00 .
Shake a second time after the block appears to be frozen.
Keep on liquid nitrogen, until TRIzol is added.
Add 1 mL of TRIzol solution.
Note
Before opening the tubes, tapping the tube on the bottom on the bench will empty most of the leaf tissue that is in the lid from the shaking process.
If it does not move, you can use the vortexer with the solution added to force it out of the lid.
Note
Optional adjustment is to add 50 µL to 100 µL of 20 % Sarkosyl to each sample with the TRIzol.
After each tube has had the TRIzol solution added, vortex immediately, both the top and bottom of the tube, until all tissue is hydrated. Vortexing for 00:02:00 can be common.
Then place tube on ice, and do this sequentially until you have all 24 tubes finished.
Once the batch is ready, incubate at Room temperature for 00:05:00 .
Centrifuge at 12000 x g for 00:10:00 at 4 °C .
Pipette aqueous solution to a new 1.7 ml tube (will be900 µL to 800 µL ).
Note
If Sarkosyl is used, be aware it will be a thick, viscous layer at the interface. Try not to pull any into the aqueous layer.
Add 200 µL of 100 % chloroform to each tube.
Note
Do not change this volume or more protein will be forced into the aqueous layer.
Vortex for 00:00:10 –00:00:15 .
Note
Solution should be milky colored.
Incubate at Room temperature for 00:10:00 .
Centrifuge at 12000 x g for 00:15:00 at 4 °C .
Note
Be careful not to disturb layers.
Remove upper aqueous layer (should be clear), 500 µL to 700 µL , and put in new 1.7 ml tube.
If you suspect the sample does not look “clean” or if you had used the Sarkosyl addition, repeat steps 16.1 to 16.5 (i.e. the chloroform step).
Precipitate and pellet the nucleic acids by adding500 µL of isopropanol.
Mix by inverting the rack.
Incubate at Room temperature for 00:10:00 .
Centrifuge at 12000 x g for 00:15:00 at 4 °C .
Pour off supernatant as waste.
Add 1000 mL of 75 % ethanol to tube with pellet.
Vortex until the pellet is loose.
Centrifuge at 8900 x g for 00:05:00 at 4 °C .
Pour off ethanol into beaker and tap tube on tissue to pull as much ethanol off as you can.
Centrifuge the “empty” tube for 00:02:00 at 4 °C .
Using pipette, pull off excess ethanol collected at the bottom of the tube.
Note
Final pellet should be clear. If white, then may still have salts and you can repeat the ethanol wash a second time, however, the DNA removal step seems to also remove salt contamination.
Let the pellet dry for 00:02:00 at Room temperature , but no more than 00:10:00 .
Re-dissolve in 50 µL of RNase-free water.
Note
If the RNA is pure, it should be instant.
To aid in dissolution, incubate at 55 °C for 00:10:00 in water bath.
Note
Never incubate longer than 00:10:00 at this temperature. Also never increase the temperature, as this can cause RNA degradation. If pellet does not dissolve immediately, store at 4 °C overnight (or until dissolved). However, the best samples dissolve with no trouble.
Vortex gently when finished.
Check on nanodrop for concentration.
Note
Samples should be diluted to be less than 200 ng/μl before proceeding to the DNAase steps. However, we have done the following steps with samples that are up to 700 ng/μl and had success. We suggest only diluting when you have 2000 ng/μl or 3000 ng/μl of RNA.
Removal of DNA using Turbo DNA-free kit by adding 0.1 volume of 10X Turbo DNasebuffer (usually 5 µL if no dilution of RNA was made) and add 1 µL of Turbo DNase to theRNA (always only 1 µL ) and mix gently.
Incubate at37 °C while shaking on the orbiter inside the incubation oven for 00:30:00 .
Add resuspended DNase Inactivation Reagent (typically 0.1 volume; 5 µL if no dilution of RNA was made) and mix well (vortex very briefly).
Incubate at Room temperature for 00:02:00 , vortexing occasionally.
Centrifuge at 10000 x g for 00:01:30 at 4 °C .
Transfer to a new tube.
Measure RNA with nanodrop again.
Note
100 ng/μl is ideal, but there will most likely be more. Expect some loss from theTurbo Kit. Also, if the first time the spectra appeared contaminated, this step may have cleaned it some. The OD 260/280 ratio should be 1.8 to 2.2 (not less than 1.6), in order to get good transcriptome library construction.