TITLE: S. palustris leaves sampling and RNA extraction 
AUTHORS: Maria Stefanie Dwiyanti

[DESCRIPTION]
This is a protocol for leaf sampling of S. palustris in natural habitats as well as RNA extraction method using CTAB+PVP buffer.
The extraction will require 3 days.

[BEFORE_START]
1. Confirm the SDS (Safety Data Sheet) for safety warnings and hazards for all reagents.
2. Use fumehood when working with phenol:chloroform:isoamylalcohol (PCI) and chloroform:isoamylalcohol (CI).
3. Follow PCI and CI disposal instruction.
4. Use gloves during RNA extraction
5. Wipe equipments with 70% ethanol or RNAse removal spray

[STEPS]
SECTION: Collecting S. palustris from field
1. Prepare RNAlater solution in 50 ml falcon tubes. 

SECTION: Collecting S. palustris from field
2. Choose young leaves without fiber. 
 
Cut leaves into small pieces into the RNAlater solution. Proportion of leaves and RNAlater solution should be maximum 1:5.

 

SECTION: Collecting S. palustris from field
3. Store 50 ml tubes inside ice box/ice packs for shipment.
SECTION: Collecting S. palustris from field
4. Leaves in RNAlater storage can be stored at 4°C up to 1 month. After that, leaves will show reddish color. It is difficult to extract RNA from reddish color leaves.
SECTION: RNA extraction
5. Warm CTAB extraction buffer to 65°C (in water bath or beaker with warm water).
SECTION: RNA extraction
6.  Add leaf materials into mortar. Wipe RNAlater left on leaves using kimwipe.

 

SECTION: RNA extraction
7. Add liquid nitrogen to mortar. Quickly crush plant material to fine powder. Keep adding liquid nitrogen while crushing, to keep plant material frozen. 
SECTION: RNA extraction
8. Add 1 spatula of sample powder to 1 ml warm CTAB buffer in 2 ml tubes. Vortex to mix the material and buffer, make sure no clumps. Add more CTAB buffer if needed.
9. Add 700 µL chloroform:isoamylalcohol (24:1). Vortex to mix.
10. Centrifuge at 15,000 rpm, 10 min, 4C
12. Repeat step 9-11  twice (total 3 times). At this stage the upper phase should be greenish-clear color.
13. Add 500 µL of 8 M LiCl, mix by inverting tubes several times.
14. Prepare ice in a styrofoam box. Put tubes in ice and store overnight at 4℃ (ideally 12-15 hours, before solution turns red)
15. Centrifuge at 15,000 rpm, 45 min, 4℃. (Thaw DNAseI buffer while waiting).
16. White-transparent pellet should appear at bottom. Discard supernatant using pipette.
17. Add 80% cold ethanol 50 µL.
18. Centrifuge 15,000 rpm, 5 min, 4℃.
19. Discard 80% ethanol by pipette.
20. Dissolve pellet in 270 µL DNase-RNAse free water. Pellet from 6 tubes can be combined into 1 tube.
21. Add 27 µL of 10×DNase I buffer to solution. Add also 2 µL of DNase I (RNase free) and 1 µL of RNase inhibitor.
22. Incubate at 37℃、30 minutes.
23. Add 300 µl of phenol:chloroform:isoamylalcohol (25:24:1), invert tubes to mix.
24. Centrifuge 15,000 rpm, 3 min, 4℃.
25. Transfer supernatant to new 1.5 ml tube, add 300 µL of chloroform:isoamylalcohol invert tubes to mix.
26. Centrifuge 15,000 rpm, 3 min, 4℃.
27. Transfer supernatant to new 1.5 ml tube, 30 µL of 3M NaOAc and 700 µl of 99.5% ethanol (4°C), mix well by inverting tubes.
28. Store in -30℃, overnight.
29. Centrifuge 15,000 rpm, 45 min, 4℃.
30. Discard supernatant using pipette. White-transparent pellet should appear at the bottom of tube.
31. Add 100 µL of 80％ ethanol (4℃),
32. Centrifuge 15,000 rpm, 3 min, 4℃ . 
34. Add 50 µL of water to dissolve pellet.
35. Quantify with Nanodrop and check quality using gel electrophoresis
36. Store RNA at -80°C.
11. Transfer upper phase to new 1.5 ml tube. 
33. Discard solution using pipette, open tubes on ice to dry pellet (a few minutes).