TITLE: Plant RNA purification using TRIzol (TRI reagent)
AUTHORS: Diep R Ganguly

[DESCRIPTION]
Extraction of total RNA from plant tissue using TRIzol (or TRI reagent) followed by DNA nuclease treatment.

[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.

[GUIDELINES]
TRI reagent and chloroform are hazardous - handle with care, operate in fume hood, wear vinly gloves and safety glasses.

[STEPS]
SECTION: RNA purification
1. Collect 50-100 mg of plant tissue and freeze immediately in liquid N2.
SECTION: RNA purification
3. 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).
SECTION: RNA purification
5. Centrifuge at 14,000 rcf for 10 minutes at 4°C.
SECTION: RNA purification
7. 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.
SECTION: RNA purification
9. Add equal volume of 100% isopropanol, then mix by inverting tubes ~20x by hand.
SECTION: RNA purification
11. Centrifuge samples at 14,000 rcf for 20 minutes at 4°C.
SECTION: RNA purification
13. Add 1 mL of 80 % ethanol and invert tube ~10x.

SECTION: RNA purification
15. Remove supernatant, carefully since the pellet often becomes dislodged at this step.

SECTION: RNA purification
16. Air-dry pellet at room temperature for 5 minutes.

SECTION: DNA nuclease treatment and ethanol precipitation
18. 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.
SECTION: Quality control
30. Take 50-100 ng aliquot of RNA and mix 1:1 with 2X RNA loading dye (NEB).
SECTION: RNA purification
2. 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.
SECTION: RNA purification
6. Transfer the upper aqueous phase to a new microfuge tube (approx. 400-600 µL).

SECTION: RNA purification
10. Incubate at -20°C for 1 hour. Alternatively, incubate overnight to capture small RNAs.
SECTION: RNA purification
4. 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.
SECTION: RNA purification
12. Remove the supernatant, you should observe a white pellet.
SECTION: RNA purification
14. Centrifuge samples at 10,000 rcf for 5 minutes at room temperature.
SECTION: RNA purification
17. 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).
SECTION: RNA purification
8. (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).
19. Add 10 μL Buffer RDD and 2.5 μL DNase I stock solution (Qiagen RNase Free DNase Set) and mix with gentle pipetting.
20. Incubate at room temperature for 5-10 minutes.
21. Add 500 μL of 100% ethanol.
22. (Optional) Add 3 μL glycogen or GlycoBlue, and 10 μL NaOAc (pH 5.5) to aid RNA precipitation.
23. Mix by gentle inversion.
24. Incubate samples for at least 1 hour at -20 °C (overnight, if purifying small RNAs).
25. Centrifuge at 14,000 rcf at 4 °C for 20 minutes. 
26. Remove supernatant and rinse pellet with 1 mL of 80% ethanol.
27. Centrifuge at 10,000 rcf for 5 minutes at 4 °C. 
28. Remove supernatant without disturbing pellet and air-dry for 2 minutes. 
29. 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).
SECTION: Quality control
32. Load and run samples on a 1% agarose TBE gel. 
SECTION: Quality control
31. Incubate RNA at 65 °C for 5 minutes.
SECTION: Quality control
33. Nanodrop RNA to check for purity and quantity.