TITLE: RNA Isolation from Plant Tissue Protocol 7: pBIOZOL-LiCl Method
AUTHORS: 

[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 cold () pBIOZOL reagent for up to  gram of frozen, ground tissue.
1.3 ml
4 °C
100 mg
?. Incubate the tube for  at .
0 Room temperature
Lay the tube down horizontally to maximize surface area during RNA extraction.
?. Centrifuge at  for .
?. Transfer the supernatant to a new 1.5 ml RNase-free tube.
?. Add  of  NaAc (pH 4.2) to the extract.
50 µl
?. Centrifuge the mixture at  for  at  to separate the phases.
4 °C
?. Add to the aqueous phase 1/3 volume of  LiCl.
?. Centrifuge the mixture at  for  at >.
4 °C
?. Decant the supernatant, taking care not to lose the pellet.
?. Centrifuge at  for  at >.
4 °C
?. Repeat the previous two steps.
?. Add  RNase-free water to dissolve the RNA pellet.
50 µl
?. Add SSTE buffer to RNA to a total volume of .
600 µl
?. Vortex the tube until the phases mix and appears cloudy.
?. Centrifuge at  for  in a microcentrifuge.
?. Transfer the top, aqueous phase to a new 1.5 ml RNase-free tube.
?. Vortex the tube until the phases mix and appear cloudy.
?. Centrifuge at  for .
?. Transfer the top aqueous phase to a new 1.5 ml RNase-free tube.
?. Centrifuge at  for  at >.
4 °C
?. Add  of 75 % ethanol to the pellet.
1 ml
?. Centrifuge at  for  at .
4 °C
?. Repeat step 18 and 19.
?. Open cap and air-dry the pellet no more than .
?. Add  RNase-free water to dissolve the pellet.
30 µl
?. Before library construction, treat RNA with DNase I.
?. Mix by briefly vortexing or flicking the bottom of the tube until the sample is thoroughly suspended.
?. Tap tube to mix.
?. Then add  NaCl and  chloroform.
100 µl
300 µl
?. Vortex vigorously.
?. Transfer the top aqueous phase (about –) to a new 1.5 ml RNase-free tube.
400 µl
500 µl
?. Mix and let stand at  overnight.
4 °C
?. Add  of 75 % ethanol to the pellet.
1 ml
?. Stand the tube at  for .
0 Room temperature
Pellet may be difficult to see.
?. Decant the liquid carefully, taking care not to lose the pellet.
?. Briefly centrifuge to collect the residual liquid and remove it with a pipette.
?. Pipette the water up and down over the pellet to dissolve the RNA..
If you extract more than   plant tissues, combine different extractions to one tube.
?. Then add equal volume of 25:24:1 acid phenol:chloroform:isoamyl alcohol to the tube.
?. 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 to the aqueous phase 2 volumes of 100 % ethanol, 1/10 volume of  NaAc (pH 5.2) and  glycogen.
2 µl
?. Invert tube to mix.
?. Store at  for .
-20 °C
?. Decant the supernatant carefully to avoid losing the pellet.
?. Incubate 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.