TITLE: RNA isolation protocol from unwilling plant tissue
AUTHORS: Samuel Parra

[DESCRIPTION]
This protocol is intended for HIGH quality RNA extraction from plan tissues 
We used this protocol after several attempts with Trizol, Qiagen RNeasy plant kit and other protocols with no ressults.
This procedure redered us between 300 to 1000 ng with good RNA

[STEPS]
SECTION: RNA isolation protocol
1. This protocol is an adaptation of https://doi.org/10.1007/BF02670468 designed for extraction of RNA from pine needles with high polyphenolic compounds or high sugar content.

For all buffers use DEPC treated water or Nuclease free ddH2O


SECTION: RNA isolation protocol
14. Reagents

SECTION: RNA isolation protocol
14. For al buffers use DEPC treated water or Nuclease free ddH20
 Extraction buffer:
 2% CTAB (hexadecyltrimethylammonium bromide)
 2% PVP (polyvinylpyrrolidinone K 30)
 100 mM Tris-HCl (pH 8.0)
 25 mM EDTA
 2.0 M NaC1
 0.5g/L spermidine
 (mix and autoclave) or microweave heat to dissolve the components, do not boil!
 2% [3-mercaptoethanol (add just before)
SECTION: RNA isolation protocol
2. Extraction buffer:
 2% CTAB (hexadecyltrimethylammonium bromide)
 2% PVP (polyvinylpyrrolidinone K 30)
 100 mM Tris-HCl (pH 8.0)
 25 mM EDTA
 2.0 M NaC1
 0.5g/L spermidine
 (mix and autoclave) Alt. microweave heat
 2% [B-mercaptoethanol] (add just before )

Ethanol 100%
LiCl2 10M
Chloroform:IAA 24:1

1.5 mL tubes
2 mL tubes

SSTE buffer:
1M NaCl
0.5% SDS
10mM TrisHCl (pH 8)
1mM EDTA (pH 8)

SECTION: Procedure, Extraction
3. For 200 mg sample use3 mLof extraction buffer.
Grind the sample with liquid nitrogen until to a fine powder. Add the 3 ml of Extraction buffer, the frozen paste will become softener in time, use the cold paste to finish grinding any remanent plant tissue. 
Separate the mixture in 3 separated 2mL tubes 
Keep the tubes on ice
on ice 

SECTION: Procedure, Extraction
4. Add an equal volumen of ChCl:IAA and vortex x 30 sec
Room temperature
Extract the WHOLE aquose phase, do not leave any of it from each tube.
ignore any coloration of the aqueus phase
*Optional : Gycogen can be added at this point
Transfer the phase to 1.5 ml tubes and add 1/4 volume of 10M LiCl and mix by inverting

SECTION: RNA cleaning
6. 13000 rpm, 10 min 
Centrifuge 10m at 13000 at RT, discard the liquid, mind the pellet!

SECTION: RNA cleaning
7. *If the SSTE buffer is below 25°C SDS on it will form flecks, warm untl sds is completely dissolved
Dissolve the pellet with500 µL of SSTE buffer. 
Join the pellet from the 3 initial tubes using the same 500 ul of SSTE.
SECTION: Procedure, Extraction
5. Precipitate overnight at 4°C4 °C 
Phases can be formed due to cold SDS in the buffers, ignore every phase or precipitation and go on.
SECTION: RNA cleaning
8. Extract with an equal volumen of CHCl:IAA by vortexing for 30 sec
13000 rpm, 10 min Centrifuge at 13000 rpm x 10m at RT

SECTION: RNA cleaning
9. Recover the upper phase completely, do not leave ANY traces, a white smudge between  the phases must be retrieved. dont worry if SOME chcl2 is tranfered.
Add 2 volumes of cold ETOH100% and precipitate for at least 120 min at -20 °C

SECTION: RNA cleaning
10. After precipitation centrifuge at 13000 rpm, 10 min, 4 °C
Discard the ethanol, DRY the rna pellet (might be invisible!)
Resuspend the pellet on 30-50 µL of ddH2O nuclease free or TE buffer