TITLE: RNA Isolation from Plant Tissue Protocol 3: CTAB-PVP Method
AUTHORS: Beijing Genomics Institute

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Beijing Genomics Institute</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 5 ml tube.
200 mg
500 mg
3 ml
?. Vortex the tube until the tissue is mixed with the buffer.
?. Incubate the tube at  for  (min), vortexing briefly () every 2–3 min during the incubation.
65 °C
?. Aliquot the mixture into four 2 ml RNase free tubes,  each tube.
1 ml
?. Spin the tube at  for  in a microcentrifuge.
All of the insoluble matter should form a pellet at the bottom of the tube.
When used for algae the centrifugation was performed at  .
?. Pour the supernatant into a new 2 ml RNase free tube.
When used for algae, 15 ml tubes were used.
?. Add equal volume of 24:1 chloroform:isoamyl alcohol to fill the tube.
?. Vortex tubes until the phases mix and appear cloudy.
?. Spin the tubes at  for  in a microcentrifuge.
When used for algae the centrifugation was performed at  .
?. Transfer the upper, aqueous phase to new 2 ml RNase free tubes.
When used for algae, 15 ml tubes were used.
?. Transfer the upper, aqueous phase to new 2 ml RNase free tubes.
?. Spin the tubes at  for  in a microcentrifuge.
?. Add  75 % ethanol to the pellet.
1 ml
?. Spin the tube at maximum speed (>) for  in a microcentrifuge.
?. Repeat steps 14 and 15 one more time.
?. Open cap and air-dry the pellet.
?. Add  RNase free water to dissolve the pellet.
30 µl
?. Combine all 4 tubes into 1 tube.
?. Add equal volume of 25:24:1 acid phenol:chloroform:isoamyl alcohol to the tube.
?. Vortex the tubes until the phases mix and appear cloudy.
?. Spin the tube at  for .
?. Transfer the upper, aqueous phase to a new 2 ml RNase free tube.
?. Vortex the tubes until the phases mix and appear cloudy.
?. Spin the tube at  for  in a microcentrifuge.
?. Transfer the upper, aqueous phase to a new 2 ml RNase free tube.
?. Spin the tube at  for  in a microcentrifuge.
?. Add  75 % cooled ethanol to the pellet.
1 ml
?. Centrifuge at  for  at .
4 °C
?. Wash the pellet twice with cooled 75 % DEPC-ethanol.
?. Add  RNase-free water to dissolve the pellet.
30 µl
?. Treat RNA with DNase I as per supplier’s protocols.
?. Repeat step 7 to 9 one more time.
?. Incubate at – for .
20 °C
25 °C
?. Add 1/3 volume of  LiCl to each tube.
?. Mix and let stand at  for – or overnight to precipitate RNA.
4 °C
?. Decant the supernatant, taking care not to lose the pellet.
?. Decant the supernatant carefully.
?. Then add SSTE buffer to each tube.
70 µl
?. Then incubate at  for .
20 °C
?. Add equal volume of 24:1 chloroform:isoamyl alcohol to the tube.
?. Then incubate at  for .
20 °C
?. Add 2 volumes of cooled 100 % ethanol, 1/10 volumes of NaAc (pH 5.2) and  glycogen.
2 µl
?. Mix and incubate at  for .
-20 °C
?. Then decant the supernatant, taking care not to lose the pellet.
?. Leave at  for .
20 °C
?. Decant the liquid carefully, taking care not to lose the pellet.
?. Briefly centrifuge to collect the residual liquid and remove it with a pipette.
?. Open cap and air-dry the pellet.