TITLE: RNA Isolation from Plant Tissue Protocol 13: Trizol/RNAqueous Midi-Kit
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Megan Rolf and Toni M. Kutchan</div><div class = "text-block">This protocol is based on a combination of two methods: The Trizol method described by</div><div class = "text-block"><span>Chomczynski and Sacchi</span><span style = "vertical-align:super;">4</span><span> and the Ambion® RNAqueous®-Midi Kit (Life Technologies, Carlsbad,</span></div><div class = "text-block">CA), with minor modifications.</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 class = "text-block"><span style = "vertical-align:super;">4</span><span>Chomczynski, P. & Sacchi, N. Single‐step method of RNA isolation by acid guanidinium thiocyanatephenol‐chloroform extraction. Analytical Biochemistry 163, 156‐159 (1987).</span></div></div>

[STEPS]
?. Quickly weigh out  of frozen tissue and place in a pre-chilled mortar containing liquid nitrogen.
3 g
?. Grind the sample into fine powder without allowing it to thaw.
?. Add the sample into a 50 ml centrifuge tube containing  of extraction buffer (10 ml buffer/g tissue).
30 ml
?. Vortex for , then incubate at  for .
0 Room temperature
?. Add  of chloroform (2 ml chloroform/g tissue) and shake vigorously for at least .
6 ml
?. Centrifuge at  for  at .
4 °C
?. Move supernatant to new 50 ml tube. Add one volume isopropanol (about ) to precipitate RNA.
22.5 ml
?. Invert gently several times. Then incubate at  for .
0 Room temperature
?. Centrifuge at  for  at .
4 °C
?. Wash pellet with  of 70 % ethanol. (1/2)
45 ml
?. Air dry pellet for  (if not completely dry, it’s still okay to move on to the next step).
?. Dissolve the pellet in  of lysis/binding Solution.
The following steps use the RNAqueous Midi Kit from Ambion (AM1911).
5 ml
Heating at   and vortexing will help dissolve pellet.
?. Heat  of elution solution to  (for later use).
4.5 ml
100 °C
Use a 17 X 100 mm round bottom sterile polypropylene tube with loose-fitting dual position cap.
?. Add  of 64 % ethanol to RNA in lysis/binding solution.
5 ml
?. Remove needle and attach filter unit. Slowly push the lysate/ethanol mixture through.
Often times the filter gets clogged. There are tips in the kit manual for dealing with this but we found these did not help. We tried to get the solution through one filter even with some intense pressure. Sometimes two filters were required and the following wash/elution steps were performed on both filters.
?. After filtering the solution, force air through using a clean 10 ml syringe until no more white foam is expelled (at least 3 or 4 times).
?. Wash with 100 % volume of Wash Solution #1 (using syringe). Use a clean 18 g needle to draw up solution.
?. Force air through a few times again.
?. Wash with 70 % volume Wash Solution #2/3. Repeat once using syringe.
?. Force air through again until no more water droplets or fine spray can be seen.
?. Elute at least 2 times into a 2 ml tube (only use  at a time, so elute three times per tube to get about 1.5 ml total) using  Elution Solution and a sterile 3 ml syringe.
500 µl
100 °C
?. LiCl precipitate each sample using the LiCl provided in the kit.
?. DNase Treatment using the TURBO DNA-free kit from ambion (AM1907)
?. LiCl precipitate the supernatant again (same as above).
The final resuspension volume should be between  –  using RNase free H2O.
?. Invert gently several times.
?. Draw into a 10 ml syringe through an 18 gauge needle.
?. Add ½ volume of LiCl.
?. Place at  for at least .
-20 °C
?. Centrifuge at maximum speed in a microcentrifuge (about ) for  at .
0 Room temperature
?. Wash pellet with  of 70 % ethanol.
1 ml
?. Centrifuge again with the same conditons for .
?. Remove supernatant and air dry pellet.
?. Resuspend in  or  RNase free H2O.
50 µl
100 µl
Heating and vortexing can help with this.
?. Add  of DNase + 0.1 volumes of 10X buffer.
1.5 µl
?. Incubate at  for .
37 °C
?. Add another  of DNase.
1.5 µl
?. Incubate at  for another .
37 °C
?. Add 0.1 volumes DNase Inactivation Reagent.
?. Incubate  at , flicking tubes every minute.
0 Room temperature
?. Centrifuge at  for .
?. Move supernatant to a new tube.
?. Then incubate at  for .
0 Room temperature
?. Centrifuge at   for  at . (1/2)
4 °C
?. Wash pellet with  of 70 % ethanol. (2/2)
45 ml
?. Centrifuge at   for  at . (2/2)
4 °C