TITLE: RNA Isolation from Plant Tissue Protocol 6: pBIOZOL and Qiagen RNeasy Plant Mini Kit Method
AUTHORS: Marc T. J. Johnson, Eric J. Carpenter, Zhijian Tian, Richard Bruskiewich, Jason N. Burris, Charlotte T. Carrigan, Mark W. Chase, Neil D. Clarke, Sarah Covshoff, Claude W. dePamphilis, Patrick P. Edger, Falicia Goh, Sean Graham, Stephan Greiner, Julian M. Hibberd, Ingrid Jordon-Thaden, Toni M. Kutchan, James Leebens-Mack, Michael Melkonian, Nicholas Miles, Henrietta Myburg, Jordan Patterson, J. Chris Pires, Paula Ralph, Megan Rolf, Rowan F. Sage, Douglas Soltis, Pamela Soltis, Dennis Stevenson, C. Neal Stewart Jr, Barbara Surek, Christina J. M. Thomsen, Juan Carlos Villarreal, Xiaolei Wu, Yong Zhang, Michael K. Deyholos, Gane Ka-Shu Wong

[DESCRIPTION]
<div class = "text-blocks"><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>) and was originally published as part of Appendix S1 of "Evaluating Methods for  Isolating Total RNA and Predicting the Success of Sequencing  Phylogenetically Diverse Plant Transcriptomes" Marc T. J. Johnson et al.  PLOS ONE, November 21, 2012.  </span><a href="https://doi.org/10.1371/journal.pone.0050226" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1371/journal.pone.0050226</span></a></div></div>

[STEPS]
?. Grind tissue to a powder in liquid nitrogen.
?. Add of cold () pBIOZOL reagent for up to  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 for  at  in a microcentrifuge at .
0 Room temperature
?. Add  of  NaCl and  chloroform.
100 µl
300 µl
?. Centrifuge at  for .
?. 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 .
?. Transfer the top aqueous phase to a new 1.5 ml RNase-free tube.
?. Pour the contents of the tube into a Qiagen mini RNA spin column (pink), until the column is almost filled with liquid.
?. Cap the tube.
?. Discard the flow-through from the collection tube.
?. Repeat the previous two steps with the same mini RNA 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.
700 µl
?. Cap the tube.
?. Discard the flow-through from the collection tube.
?. Apply  of solution RPE to the spin column.
500 µl
?. Discard the flow-through.
?. Repeat previous two steps one time.
?. Spin at maximum speed for  to remove remaining liquid from the silica membrane.
?. Transfer the spin column to a new 1.5 ml conical bottom microcentrifuge tube.
?. Add – of RNase-free water to the column.
30 µl
50 µl
?. Spin at maximum speed for  to collect RNA solution.
?. Mix by briefly vortexing or flicking the bottom of the tube until the sample is thoroughly re-suspended.
?. Transfer the supernatant to a new  RNase-free tube.
1.5 ml
?. Vortex vigorously.
?. Add an equal volume of 5:1 acid phenol:chloroform to the tube.
?. Incubate at  for .
20 °C
?. Add to the aqueous phase equal volume of 24:1 chloroform:isoamyl alcohol.
?. Vortex the tube until the phases mix and appear cloudy.
?. Then incubate at  for .
0 Room temperature
?. Add 1/2 volume of 100 % ethanol.
?. Centrifuge at  for .
The column should be empty at the end of this spin.
?. Centrifuge at  for .
The column should be empty at the end of this spin.
?. Cap the tube
?. Centrifuge at  for .
The column should be empty at the end of this spin.
?. Then let tube incubate at  for .
20 °C