TITLE: RNA Isolation from Plant Tissue Protocol 8: CTAB/Acid Phenol/Silica Membrane Method
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Michael Deyholos</div><div class = "text-block">This protocol combines elements of standard CTAB, acid phenol, and silica-membrane protocols. </div><div class = "text-block">The protocol was developed to extract total RNA from a wide range of plant species and tissues, and to do so in the smallest volume possible to yield >20 μg of total RNA using as little as 50 mg of fresh tissue. Maintaining a small extraction volume also allows for many samples to be processed in parallel in microcentrifuge tubes; </div><div class = "text-block">at least 12 samples can be processed in 3–4 hours. </div><div class = "text-block">The protocol has been used successfully with dozens of species, including tissues rich in polysaccharides and/or secondary metabolites. There are four organic extractions in total (three chloroform and one phenol:chloroform). </div><div class = "text-block">For many species/tissues, the full set of chloroform extractions in the order specified is required to maximize RNA purity and to prevent phase inversion during the acid phenol extraction.</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]
?. Grind tissue to a powder in liquid nitrogen.
?. Add– of ground, frozen tissue to  of pre-heated extraction buffer in a 2 ml microcentrifuge tube.
400 mg
600 mg
1.4 ml
?. Vortex the tube until the tissue is mixed with the buffer.
To facilitate mixing, you may have to invert the tube on the vortex, and/or heat it briefly in a   water bath.
?. Incubate the tube at  for –, vortexing briefly (15 seconds) twice during the incubation.
65 °C
?. Spin the tube at maximum speed (>) for  in a microcentrifuge.
All of the insoluble matter should form a pellet at the bottom of the tube.
?. Pour the supernatant into a new 2 ml tube.
?. [Solvent Extraction #1]
Add enough 24:1 chloroform:isoamyl alcohol to fill the tube.
?. [Solvent Extraction #1]
Vortex the tube for  or until the phases mix and appear cloudy.
?. [Solvent Extraction #1]
Spin the tube at maximum speed (>) for  in a microcentrifuge.
Most of the chlorophyll will be dissolved in the lower, organic phase.
?. [Solvent Extraction #1]
Transfer the upper, aqueous phase to a new 2 ml tube, using a disposable pipette.
Avoid transferring any of the material (usually a white precipitate) from the boundary between the phases.
?. [Solvent Extraction #2]
Add 24:1 chloroform:isoamyl alcohol to the tube containing the aqueous phase (this should be at least  of 24:1 chloroform:isoamyl).
900 µl
?. [Solvent Extraction #2]
Vortex the tube for  or until the phases mix and appear cloudy.
?. [Solvent Extraction #2]
Spin the tube at maximum speed (>) for  in a microcentrifuge.
?. [Solvent Extraction #2]
Transfer the upper, aqueous phase to a new 2 ml tube, using a disposable pipette.
Avoid transferring any of the material (usually a white precipitate) from the boundary between the phases.
?. [Solvent Extraction #3]
Add  5:1 phenol:chlorofom pH 4.5 to the tube containing the aqueous phase.
1 ml
?. [Solvent Extraction #3]
Vortex the tube for  or until the phases mix and appear cloudy.
?. [Solvent Extraction #3]
Spin the tube at maximum speed (>) for  in a microcentrifuge.
?. [Solvent Extraction #3]
Transfer the upper, aqueous phase to a new 2 ml tube, using a disposable pipette.
Avoid transferring any of the material (usually a white precipitate) from the boundary between the phases.
?. [Solvent Extraction #4]
Add 24:1 chloroform:isoamyl alcohol to the tube containing the aqueous phase (this should be at least  of 24:1 chloroform:isoamyl).
900 µl
?. [Solvent Extraction #4]
Vortex the tube for  or until the phases mix and appear cloudy.
?. [Solvent Extraction #4]
Spin the tube at maximum speed (>) for  in a microcentrifuge.
?. [Solvent Extraction #4]
Transfer the upper, aqueous phase to a new 2 ml tube, using a disposable pipette.
Avoid transferring any of the material (usually a white precipitate) from the boundary between the phases.
?. Estimate the volume of the aqueous phase based on the markings on the tube.
?. Estimate the new total volume in the tube.
?. Pour the contents of the tube into a Qiagen miniRNA spin column (pink), until the column is almost filled with liquid.
?. Cap the tube and spin for  at >.
The column should be empty at the end of this spin.
?. Discard the flow-through from the collection tube.
?. Repeat the previous two steps with the same miniRNA spin column, until all of the liquid in the tube(s) has been passed through the column.
The nucleic acid is now bound to the silica membrane in the spin column.
?. Apply  of solution RW1 to the spin column.
350 µl
?. Cap the tube and spin for  at >.
The column should be empty at the end of this spin.
?. Discard the flow-through from the collection tube.
?. Apply  of DNase digestion solution to the membrane of the spin column.
80 µl
?. Incubate at room temperature for .
?. Apply  of solution RW1 to the spin column.
350 µl
?. Cap the tube and spin for  at >.
The column should be empty at the end of this spin.
?. Discard the flow-through from the collection tube.
?. Apply  of solution RPE to the spin column.
500 µl
?. Cap the tube and spin for  at >.
The column should be empty at the end of this spin.
?. Discard the flow-through from the collection tube.
?. Apply  of solution RPE to the spin column.
500 µl
?. Cap the tube and spin for  at >.
The column should be empty at the end of this spin.
?. Discard the flow-through from the collection tube.
?. Transfer the spin column to a new collection tube.
?. Transfer the spin column to a new 1.5 ml conical bottom microcentrifuge tube
?. Add  of RNase-free water to the column.
44 µl
?. Spin at maximum speed for  to elute.
?. Add at least 0.5 volumes of solution RLT, and mix by briefly shaking.
?. Add 0.5 volumes of 95–100 % ethanol.
?. Mix by briefly shaking.
?. Spin at maximum speed for  to remove remaining liquid from the silica membrane.