TITLE: RNA Extraction Protocol for Leaves with High Content of Secondary Metabolites
AUTHORS: Wesley Elias Bhering Barrios, Débora Gonçalves Gouveia

[DESCRIPTION]
Protocol used for extraction of total RNA from plant material rich in secondary metabolites.

[BEFORE_START]
Important details:
*Sterilize everything before and ensure that all material is suitable for RNA extraction (molecular grade).
*Aliquot the Trizol, Chloroform and Ethanol 99% for Mol. Bio.  in 50 mL Falcons, and leave them cold before starting the extraction (-20ºC).
*Leave all 1.5 mL tubes ready for appropriate tube changes (2 changes).
**Make a maximum of 12-16 tubes per shift (up to 24 with two people).
*Samples taken from the -80 °C freezer should be kept in a bottle of liquid nitrogen. When adding the Trizol, we must take the samples out of the bottle and immediately add the Trizol to avoid enzymatic degradation of the RNA.
**Add as soon as possible the 500 uL of Trizol.
***Before starting, read the protocol and ensure that the necessary volume of each reagent is available.
*Before starting, place a microtube rack that fits all your samples in the -20 °C freezer.
***75% ethanol should be prepared with NEW DEPC H2O and Ethanol for Molecular Biology 200 Proof >99.45%.



[STEPS]
SECTION: Procedure
1. Collect the plant material and freeze it immediately in liquid nitrogen;
SECTION: Procedure
2. Macerate and transfer approximately 20 to 30 mg (max. 50 mg) to a 1.5 mL microtube;
SECTION: Procedure
3. Add 500 uL of TRIzol to every 3 tubes at once (with two people, otherwise from tube to tube if alone);
SECTION: Procedure
4. Homogenize by vortexing briefly until the solution turns brown (5 seconds);
SECTION: Procedure
3.1. Add in each tube 30mg of PVPP (2 full aliquot spoons);
SECTION: Procedure
5. Incubate at room temperature for 5 minutes (dissociate ribonucleoproteins);
SECTION: Procedure
6. Add 100 uL of cold Chloroform (-20ºC);
SECTION: Procedure
7. Vortex vigorously for 15 seconds;
SECTION: Procedure
8. Incubate in the thermomixer at 23°C (room T), 700 RPM for 15 min;
SECTION: Procedure
9. Centrifuge at 12,000 RPM for 15 min at 4°C to separate the two phases: organic and aqueous (RNA);
SECTION: Procedure
10. Collect the aqueous phase (200 to 300uL - as much as you can get) and transfer to new, labeled microtubes (1.5 mL) (we collected 250uL for safety);

    *Don't touch the middle phase with the pipette tips.
SECTION: Procedure
11. Add 1 volume of chloroform (200 to 300 uL) - 250 uL in our case;
SECTION: Procedure
12. Invert the tubes 20x using a microtube rack, or gently vortex the tubes for 2 seconds;
SECTION: Procedure
13. Incubate in the thermomixer at 23°C (room T), 700 RPM for 10 minutes;
SECTION: Procedure
14. Centrifuge at 13,000 RPM for 10 minutes at 4°C;
SECTION: Procedure
15. Collect 100 to 200 uL of the supernatant (we collected 175 uL for safety) and transfer to new, labeled microtubes (1.5 mL);
SECTION: Procedure
16. Add 1 volume of ice-cold isopropanol (175 uL) - (leave at -20°C until the time of application). 
*Use only Isopropanol Molecular Biology Grade - 200 Proof (>99.45%);
SECTION: Procedure
17. Invert the tubes for 30 seconds using a microtube rack or gently vortex the tubes for 2 seconds;
SECTION: Procedure
18. Incubate for 30 min at -80 °C (ultra freezer) in a new rack previously placed at -80 °C or leave overnight at -20 °C;
SECTION: Procedure
19. Centrifuge at 13,000 RPM for 15 minutes at 4°C;
SECTION: Procedure
20. Carefully discard the isopropanol with the aid of pipette tips;
SECTION: Procedure
21. Add 500 uL of 75% ethanol to wash the pellet and rehydrate the RNA 
- Use Molecular Biology Grade Ethanol - 200 proof to prepare this solution;
    
*We must add the 75% EtOH solution directly to the RNA pellet, homogenizing by vortexing (lightly tapping the vortex, <1 second), just to WASH it, to do this, centrifuge the tubes with the hinge facing away from the rotor, the pellet will be at the bottom of the tube in the exact direction of the hinge.
SECTION: Procedure
22. Centrifuge at 13,000 RPM for 10 minutes at 4°C;
SECTION: Procedure
23. Carefully dispose of the ethanol with the aid of a pipette tip;
SECTION: Procedure
24. Repeat the last 3 steps (21,22 and 23);

    *Spin the microtubes and remove the excess ethanol using pipette tips, taking care not to aspirate the pellet.
SECTION: Procedure
25. Place the open tubes in the laminar flow cabinet with the glass closed at room temperature for 5 minutes (pay attention to which location the airflow is strongest, and position the rack at this location);
SECTION: Procedure
26. Resuspend in 30 uL of autoclaved Milli-Q Water treated with 0.1% DEPC at 60 °C;
    *Add water to all tubes immediately after leaving the cabin;
    *Homogenizing with up & down (setting the pipette at 25 uL so that no bubbles form, or homogenizing carefully at 30 uL) for 30 seconds per sample.
    *This step takes 1 minute per tube!
SECTION: Procedure
27. Treat with DNase - gDNA RNA CleanUp Protocol;
SECTION: Procedure
28. Run 1.5% chlorine-agarose gel (3 uL sample + 2 ul loading buffer 6X (Gel Red at 9X) - 80 V - 80 min);
    ***The electrophoresis tank should be cleaned well with distilled water and fresh running buffer should be added, and the gel polymerization tray should be cleaned well, and the gel should be made with fresh buffer to avoid RNA degradation.
***Chlorine gel: Use 5% commercial bleach added to TAE 1X buffer.
SECTION: Procedure
30. Store the samples in the ultra-freezer (-80 ºC) for up to 6 months.
SECTION: Procedure
29. Quantify RNA purity and concentration in the Nanodrop or Qubit;
    -Pure RNA has 260/280 ratio > 1.8 and 260/230 ratio > 1.6.
For RNAseq, Microarray or more sensitive downstream RNA applications, quantify RNA integrity with Bioanalyzer 2100 (RNA 6000 kits) or Tapestation 2200; always using Plant RNA Algorithm (refer to the software version manual)