TITLE: Isolation of high quality RNA from tree leaves using Polyclar in the Spectrum Plant Total RNA Kit
AUTHORS: Tobias Bruegmann, Franziska Orgel, Susanne Jelkmann, Hilke Schroeder

[DESCRIPTION]
This protocol is based on the use of the Spectrum Plant Total RNA Kit from Sigma. The protocol has been adapted for the isolation of RNA from plant tissue of trees. It was tested with good results for pedunculate oak (Quercus robur), European beech (Fagus sylvatica), and poplar hybrids (Populus tremula × P. alba).

[BEFORE_START]
Preheat a heatblock to 56 °C. Add ethanol to Wash Buffer 2 during the first extraction according the manufacturer's guidelines.

[GUIDELINES]
When working with RNA, you should bear in mind that RNA is rapidly degraded by ubiquitous RNases. Therefore, rapid processing without unnecessary lengths is highly recommended. Substances for the removal of active RNases, e.g. "RNase-Away", should be used to clean the workplace and required utensils.

[STEPS]
SECTION: Sample preparation
1. Harvest plant material and freeze it immediately in liquid nitrogen. Alternatively, you can use frozen plant material.
SECTION: Sample preparation
2. Grind approximately 100 mg using a swing mill (2-3 x 1:15 min at 17.5 Hz) with small metal balls or using a bead ruptor (3 × 15 s at 2.1 m/s).
SECTION: Preparation of required solutions
5. Add 60 mg Polyclar per mL Lysis Solution.
SECTION: Preparation of required solutions
4. Add β-Mercaptoethanol to the Lysis Solution (10 µL β-Mercaptoethanol per mL Lysis Solution). 

SECTION: RNA isolation procedure
7. Add 500 µL Lysis Solution supplemented with Polyclar and β-Mercaptoethanol to each sample and vortex vigorously for 30 s immediately.
SECTION: RNA isolation procedure
8. Incubate for 3-5 min at 56 °C in the heatblock. During incubation you can start preparing the filtration columns (consider proper labelling to avoid mixing up samples). 
SECTION: RNA isolation procedure
9. Centrifuge for 3 min at 16,000 × g, while preparing filtration columns (blue).
SECTION: RNA isolation procedure
10. Transfer the supernatant to the filtration column and close the lid of the tube. Do not pipette any of the pellet. Small suspended particles should not pose a problem.
SECTION: RNA isolation procedure
11. Centrifuge for 1 min at max. speed (16,000 × g). Repeat this centrifugation if the lysate has not run through the column completely. Do not discard the flow-through.
SECTION: RNA isolation procedure
12. Prepare the binding columns.
SECTION: RNA isolation procedure
13. Continue with Protocol B according the manufacturer's manual: Add 250 µL Binding Solution to the filtered lysate, mix by pipetting (pipette up and down at least 5 times).
SECTION: Preparation of required solutions
6. Thaw DNase I Digestion Buffer and prepare the DNase digestion mix. Carefully mix 10 µL DNase I and 70 µL Digestion Buffer per sample. Store the digestion mix on ice until utilization.
SECTION: RNA isolation procedure
14. Pipette the mixture to the binding column and close the tube cap.
SECTION: RNA isolation procedure
15. Centrifuge for 1 min at max. speed (16,000 × g). 
SECTION: RNA isolation procedure
16. Discard the flow-through and reuse the collection tube.
SECTION: RNA isolation procedure
17. Add 300 µL of Wash Solution 1 to the column and close the cap.
SECTION: RNA isolation procedure
18. Centrifuge for 1 min max. speed (16,000 × g). 
SECTION: RNA isolation procedure
19. Discard the flow-through and reuse the collection tube.
SECTION: RNA isolation procedure
20. Pipette 80 µL of DNase I digestion mix onto the column and incubate at room temperature for at least 15 min (preferably 30 min).
SECTION: RNA isolation procedure
21. Add 500 µL of Wash Solution 1 to the column and close the cap.
SECTION: RNA isolation procedure
22. Centrifuge for 1 min max. speed (16,000 × g). 
SECTION: RNA isolation procedure
23. Discard the flow-through, and reuse the tube.
SECTION: RNA isolation procedure
24. Add 500 µL of Wash Solution 2 to the column and close the cap.
SECTION: RNA isolation procedure
25. Centrifuge for 30 s max. speed (16,000 × g). 
SECTION: RNA isolation procedure
26. Discard the flow-through and reuse the collection tube.
SECTION: RNA isolation procedure
27. Repeat the wash cycle: Add 500 µL of Wash Solution 2 to the column and close the cap.
SECTION: RNA isolation procedure
28. Centrifuge for 30 s max. speed (16,000 × g). 
SECTION: RNA isolation procedure
29. Discard the flow-through and reuse the collection tube.
SECTION: RNA isolation procedure
30. Centrifuge for 1 min max. speed (16,000 × g) to dry the column.
SECTION: RNA isolation procedure
31. Carefully place the column in a new collection tube.
SECTION: RNA isolation procedure
32. Pipette 50 µL of Elution Solution into the center of the column matrix and incubate for 1-2 min at room temperature.
SECTION: RNA isolation procedure
33. Centrifuge for 1 min max. speed (16,000 × g). 
SECTION: RNA isolation procedure
34. Place the samples on ice for immediate processing or freeze at -70°C for later use.
SECTION: Preparation of required solutions
3. Take an aliquot of the Lysis Solution according to the number of your samples and transfer this volume into a separate vessel, such as an RNase-free centrifugation tube. For your calculations: 500 µL of Lysis Solution, including the additives, will be used per sample later.