Jan 31, 2024
Version 2
Plant RNA purification using TRIzol (TRI reagent) V.2
- 1University of Pennsylvania
External link: https://doi.org/10.1073/pnas.2121362119
Protocol Citation: Diep R Ganguly 2024. Plant RNA purification using TRIzol (TRI reagent). protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq4enyvk5/v2Version created by Diep R Ganguly
Manuscript citation:
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: January 30, 2024
Last Modified: January 31, 2024
Protocol Integer ID: 94427
Abstract
Extraction of total RNA from plant tissue using TRIzol (or TRI reagent) followed by DNA nuclease treatment.
Guidelines
TRI reagent and chloroform are hazardous - handle with care, operate in fume hood, wear vinly gloves and safety glasses.
Materials
- TRIzol (or TRI reagent)
- Chloroform (or chloroform : IAA [24:1])
- Isopropanol
- 80% Ethanol
- Nuclease-free H2O (e.g. 0.01% DEPC-treated H20) or Tris-EDTA (10 mM Tris-Cl, pH 6.5, 0.1 mM EDTA)
- 2 mL safe-lock Eppendorf tubes
- 1.5 mL microcentrifuge tubes
- Tissue lyser or mortar and pestle
- RNase-Free DNase Set (Qiagen)
- RNA loading dye, 2X (NEB)
Safety warnings
TRI reagent - hazardous
Chloroform - hazardous
Ensure you read SDS documents (attached) and organise appropriate waste vessels (fume hood).
Before start
Ensure benches and equipment are RNase free.
For RNase-Free DNase Set (Qiagen): Prepare DNase I stock solution by dissolving the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNase-free water. Mix gently by inverting the vial. Divide into single use aliquots and store at -20 °C. Thawed aliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after thawing.
RNA purification
RNA purification
Collect 50-100 mg of plant tissue and freeze immediately in liquid N2.
Grind tissue to fine powder under liquid N2 using tissue lyser or mortar + pestle, then immediately add 1 mL TRI reagent (1 mL per 100 mg tissue).
Note, achieving a fine grind is critical to high yields of intact RNA.
Invert each tube by hand ~20x and incubate at room temperature for 5 minutes (DO NOT vortex samples as it may result in RNA degradation).
Add 1/5 volume of pre-mixed chloroform:isoamyl alcohol (24:1), cap tubes, shake vigorously (by hand) for 15 seconds (solution should become cloudy), then incubate at room temperature for 3 minutes.
Centrifuge at 14,000 rcf for 10 minutes at 4°C.
Transfer the upper aqueous phase to a new microfuge tube (approx. 400-600 µL).
Repeat steps 4 and 5 (approx. 300-400 µL).
Note, if you are observing buffer and salt carryover in your purified RNA (high 230 nm absorbance), reduce volume of upper-phase recovered.
(Optional) If the expected RNA concentration is 10 µg/mL, add 1/10 volume of 3M NaOAc (pH 5.5) and/or 100 µg/mL GlycoBlue (or glycogen).
Add equal volume of 100% isopropanol, then mix by inverting tubes ~20x by hand.
Incubate at -20°C for 1 hour. Alternatively, incubate overnight to capture small RNAs.
Centrifuge samples at 14,000 rcf for 20 minutes at 4°C.
Remove the supernatant, you should observe a white pellet.
Add 1 mL of 80 % ethanol and invert tube ~10x.
Centrifuge samples at 10,000 rcf for 5 minutes at room temperature.
Remove supernatant, carefully since the pellet often becomes dislodged at this step.
Air-dry pellet at room temperature for 5 minutes.
Resuspend pellet in RNase-free water (e.g. 0.01% DEPC-treated water) or Tris-EDTA (10 mM Tris-Cl, pH 6.5, 0.1 mM EDTA).
DNA nuclease treatment and ethanol precipitation
DNA nuclease treatment and ethanol precipitation
Make up volume of RNA solution to 87.5 μL with nuclease-free water. This can be performed with an aliquot or total sample from the previous step.
Add 10 μL Buffer RDD and 2.5 μL DNase I stock solution (Qiagen RNase Free DNase Set) and mix with gentle pipetting.
Incubate at room temperature for 5-10 minutes.
Add 500 μL of 100% ethanol.
(Optional) Add 3 μL glycogen or GlycoBlue, and 10 μL NaOAc (pH 5.5) to aid RNA precipitation.
Mix by gentle inversion.
Incubate samples for at least 1 hour at -20 °C (overnight, if purifying small RNAs).
Centrifuge at 14,000 rcf at 4 °C for 20 minutes.
Remove supernatant and rinse pellet with 1 mL of 80% ethanol.
Centrifuge at 10,000 rcf for 5 minutes at 4 °C.
Remove supernatant without disturbing pellet and air-dry for 2 minutes.
Resuspend pellet in RNase-free water (e.g. 0.01% DEPC-treated water) or Tris-EDTA (10 mM Tris-Cl, pH 6.5, 0.1 mM EDTA).
Quality control
Quality control
Take 50-100 ng aliquot of RNA and mix 1:1 with 2X RNA loading dye (NEB).
Incubate RNA at 65 °C for 5 minutes.
Load and run samples on a 1% agarose TBE gel.
Nanodrop RNA to check for purity and quantity.