Aug 20, 2019
RNA Isolation from Plant Tissue Protocol 13: Trizol/RNAqueous Midi-Kit
Peer-reviewed method
- GigaScience Press
External link: https://doi.org/10.1093/gigascience/giz126
Protocol Citation: Eric Carpenter: RNA Isolation from Plant Tissue Protocol 13: Trizol/RNAqueous Midi-Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.4tjgwkn
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: 25163
Keywords: RNA, RNA extraction, RNA isolation, plant tissue
Abstract
Implemented by: Megan Rolf and Toni M. Kutchan
Note
Note: (two samples from C. dePamphilis and P. Ralph only used the RNAqueous Midi-Kit)
This protocol is based on a combination of two methods: The Trizol method described by
Chomczynski and Sacchi4 and the Ambion® RNAqueous®-Midi Kit (Life Technologies, Carlsbad,
CA), with minor modifications.
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)
4Chomczynski, P. & Sacchi, N. Single‐step method of RNA isolation by acid guanidinium thiocyanatephenol‐chloroform extraction. Analytical Biochemistry 163, 156‐159 (1987).
Attachments
Materials
MATERIALS
RNAqueous™-Midi Total RNA Isolation KitThermo Fisher ScientificCatalog #AM1911
TURBO DNA-free™ KitThermo Fisher ScientificCatalog #AM1907
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Quickly weigh out 3 g of frozen tissue and place in a pre-chilled mortar containing liquid nitrogen.
Grind the sample into fine powder without allowing it to thaw.
Add the sample into a 50 ml centrifuge tube containing 30 mL of extraction buffer (10 ml buffer/g tissue).
Vortex for 00:01:00 , then incubate at Room temperature for 00:05:00 .
Add 6 mL of chloroform (2 ml chloroform/g tissue) and shake vigorously for at least 00:00:20 .
Centrifuge at 3095 x g for 00:20:00 at 4 °C .
Move supernatant to new 50 ml tube. Add one volume isopropanol (about 22.5 mL ) to precipitate RNA.
Invert gently several times. Then incubate at Room temperature for 00:10:00 .
Invert gently several times.
Then incubate at Room temperature for 00:10:00 .
Centrifuge at 3095 x g for 00:10:00 at 4 °C .
Wash pellet with 45 mL of 70 % ethanol. (1/2)
Centrifuge at 3095 x g for 00:02:00 at 4 °C . (1/2)
Wash pellet with 45 mL of 70 % ethanol. (2/2)
Centrifuge at 3095 x g for 00:02:00 at 4 °C . (2/2)
Air dry pellet for 00:05:00 (if not completely dry, it’s still okay to move on to the next step).
Note
Dissolve the pellet in 5 mL of lysis/binding Solution.
Note
Heating at 37 °C and vortexing will help dissolve pellet.
Heat 4.5 mL of elution solution to 100 °C (for later use).
Note
Use a 17 X 100 mm round bottom sterile polypropylene tube with loose-fitting dual position cap.
Add 5 mL of 64 % ethanol to RNA in lysis/binding solution.
Draw into a 10 ml syringe through an 18 gauge needle.
Remove needle and attach filter unit. Slowly push the lysate/ethanol mixture through.
Note
Often times the filter gets clogged. There are tips in the kit manual for dealing with this but we found these did not help. We tried to get the solution through one filter even with some intense pressure. Sometimes two filters were required and the following wash/elution steps were performed on both filters.
After filtering the solution, force air through using a clean 10 ml syringe until no more white foam is expelled (at least 3 or 4 times).
Wash with 100 % volume of Wash Solution #1 (using syringe). Use a clean 18 g needle to draw up solution.
Force air through a few times again.
Wash with 70 % volume Wash Solution #2/3. Repeat once using syringe.
Force air through again until no more water droplets or fine spray can be seen.
Elute at least 2 times into a 2 ml tube (only use 500 µL at a time, so elute three times per tube to get about 1.5 ml total) using 100 °C Elution Solution and a sterile 3 ml syringe.
LiCl precipitate each sample using the LiCl provided in the kit.
Add ½ volume of LiCl.
Place at -20 °C for at least 00:30:00 .
Centrifuge at maximum speed in a microcentrifuge (about 16000 x g ) for 00:15:00 at Room temperature .
Wash pellet with 1 mL of 70 % ethanol.
Centrifuge again with the same conditons for 00:05:00 .
Remove supernatant and air dry pellet.
Resuspend in 50 µL or 100 µL RNase free H2O.
Note
Heating and vortexing can help with this.
DNase Treatment using the TURBO DNA-free kit from ambion (AM1907)
Add 1.5 µL of DNase + 0.1 volumes of 10X buffer.
Incubate at 37 °C for 00:25:00 .
Add another 1.5 µL of DNase.
Incubate at 37 °C for another 00:25:00 .
Add 0.1 volumes DNase Inactivation Reagent.
Incubate 00:05:00 at Room temperature , flicking tubes every minute.
Centrifuge at 10000 x g for 00:01:30 .
Move supernatant to a new tube.
LiCl precipitate the supernatant again (same as above).
Note
The final resuspension volume should be between 50 µL –100 µL using RNase free H2O.