TITLE: RNA Isolation from Plant Tissue Protocol 14: Ambion Trizol RNA Extraction in Microcentrifuge Tubes with Turbo DNAfree Digestion
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Ingrid Jordon-Thaden and Nicholas Miles (Soltis Labs)</div><div class = "text-block">This procedure eliminates the mortar and pestle homogenization of tissues and instead grinds tissue in 2 ml microcentrifuge tubes. The method closely follows Ambion’s protocols and could be used in a 96-well format. This method worked great for species that proved to be difficult to extract with other methods (i.e. woody and aquatic plants).</div><div class = "text-block"><span>This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: </span><a href="https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6</span></a><span>) </span></div></div>

[STEPS]
?. Clean all surfaces and equipment with 20 % bleach, 95 % ethanol, and RNase Zap, and place supplies in hood.
?. Prepare sample tubes with labels.
?. Fill microcentrifuge tubes with 5 zirconia beads (if they have not been done before tissue collection).
?. Prepare aliquots of the following solutions into 50mL Falcon tubes: 75 % ethanol, 100 % chloroform, 100 % isopropanol.
?. Fill dewar with liquid N2.
?. Get samples from  and place them in the black shaker block that is sitting in a bath of liquid N2 (if already in microcentrifuge tubes), and check labels.
-80 °C
?. Prepare frozen tissue in microcentrifuge tubes from 50 ml Falcons (if tissue is not in them already).
?. Before pulverizing the frozen tissue, check each tube for beads, making sure they are easily moving within the tube.
?. Taking the black shaker block with the 24 samples, place tightly into the automatic shaker, doing this quickly as to not allow thawing.
?. Place block back into the liquid nitrogen cooler if needed to shake another .
?. Add  of TRIzol solution.
1 ml
Before opening the tubes, tapping the tube on the bottom on the bench will empty most of the leaf tissue that is in the lid from the shaking process.If it does not move, you can use the vortexer with the solution added to force it out of the lid.
Optional adjustment is to add   to   of 20 % Sarkosyl to each sample with the TRIzol.
?. After each tube has had the TRIzol solution added, vortex immediately, both the top and bottom of the tube, until all tissue is hydrated. Vortexing for  can be common.
?. Then place tube on ice, and do this sequentially until you have all 24 tubes finished.
?. Once the batch is ready, incubate at  for .
0 Room temperature
?. Centrifuge at  for  at .
4 °C
?. Pipette aqueous solution to a new 1.7 ml tube (will be to ).
900 µl
800 µl
If Sarkosyl is used, be aware it will be a thick, viscous layer at the interface. Try not to pull any into the aqueous layer.
?. Precipitate and pellet the nucleic acids by adding of isopropanol.
500 µl
?. Incubate at  for .
0 Room temperature
?. Centrifuge at  for  at .
4 °C
?. Pour off supernatant as waste.
?. Add  of 75 % ethanol to tube with pellet.
1000 ml
?. Centrifuge at  for  at .
4 °C
?. Pour off ethanol into beaker and tap tube on tissue to pull as much ethanol off as you can.
?. Centrifuge the “empty” tube for  at .
4 °C
?. Using pipette, pull off excess ethanol collected at the bottom of the tube.
Final pellet should be clear. If white, then may still have salts and you can repeat the ethanol wash a second time, however, the DNA removal step seems to also remove salt contamination.
?. Let the pellet dry for  at , but no more than .
0 Room temperature
?. Re-dissolve in  of RNase-free water.
50 µl
If the RNA is pure, it should be instant.
?. Check on nanodrop for concentration.
Samples should be diluted to be less than   before proceeding to the DNAase steps. However, we have done the following steps with samples that are up to   and had success. We suggest only diluting when you have   or   of RNA.
?. Removal of DNA using Turbo DNA-free kit by adding 0.1 volume of 10X Turbo DNasebuffer (usually  if no dilution of RNA was made) and add  of Turbo DNase to theRNA (always only ) and mix gently.
5 µl
1 µl
1 µl
?. Incubate at while shaking on the orbiter inside the incubation oven for .
37 °C
?. Add resuspended DNase Inactivation Reagent (typically 0.1 volume;  if no dilution of RNA was made) and mix well (vortex very briefly).
5 µl
?. Incubate at  for , vortexing occasionally.
0 Room temperature
?. Centrifuge at  for  at .
4 °C
?. Measure RNA with nanodrop again.
is ideal, but there will most likely be more. Expect some loss from theTurbo Kit. Also, if the first time the spectra appeared contaminated, this step may have cleaned it some. The OD 260/280 ratio should be 1.8 to 2.2 (not less than 1.6), in order to get good transcriptome library construction.
?. Fill two small coolers with liquid nitrogen (one for microcentrifuge tubes, the other for Falcon tubes).
?. Doing about 3 or 4 plants at a time, use forceps or a spatula to move  to  of tissue into the microcentrifuge tubes from the 50 ml Falcon tubes.
60 mg
100 mg
?. Get a weight for each of the samples, add more if necessary.
?. This kit requires  to  of plant tissue.
60 mg
100 mg
If you are doing aquatic plants use 2x the weight since so much of the weight is water.
?. Repeat these steps until you have finished all of the samples.
?. Shake for  (they will stay frozen).
?. Shake a second time after the block appears to be frozen.
?. Keep on liquid nitrogen, until TRIzol is added.
?. Add   of 100 % chloroform to each tube.
200 µl
Do not change this volume or more protein will be forced into the aqueous layer.
?. Vortex for –.
Solution should be milky colored.
?. Incubate at   for .
0 Room temperature
?. Centrifuge at  for  at .
4 °C
Be careful not to disturb layers.
?. Remove upper aqueous layer (should be clear),  to , and put in new 1.7 ml tube.
500 µl
700 µl
?. If you suspect the sample does not look “clean” or if you had used the Sarkosyl addition, repeat steps 16.1 to 16.5 (i.e. the chloroform step).
?. Mix by inverting the rack.
?. Vortex until the pellet is loose.
?. To aid in dissolution, incubate at  for  in water bath.
55 °C
Never incubate longer than   at this temperature. Also never increase the temperature, as this can cause RNA degradation. If pellet does not dissolve immediately, store at   overnight (or until dissolved). However, the best samples dissolve with no trouble.
?. Vortex gently when finished.
?. Transfer to a new tube.