Aug 20, 2019

Public workspaceRNA Isolation from Plant Tissue Protocol 6: pBIOZOL and Qiagen RNeasy Plant Mini Kit Method

GigaScience
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.4rfgv3n
  • Marc T. J. Johnson1,
  • Eric J. Carpenter2,
  • Zhijian Tian3,
  • Richard Bruskiewich4,
  • Jason N. Burris5,
  • Charlotte T. Carrigan6,
  • Mark W. Chase7,
  • Neil D. Clarke8,
  • Sarah Covshoff9,
  • Claude W. dePamphilis10,
  • Patrick P. Edger11,
  • Falicia Goh8,
  • Sean Graham12,
  • Stephan Greiner13,
  • Julian M. Hibberd9,
  • Ingrid Jordon-Thaden14,15,
  • Toni M. Kutchan16,
  • James Leebens-Mack6,
  • Michael Melkonian17,
  • Nicholas Miles14,15,
  • Henrietta Myburg18,
  • Jordan Patterson2,
  • J. Chris Pires11,
  • Paula Ralph10,
  • Megan Rolf16,
  • Rowan F. Sage19,
  • Douglas Soltis14,
  • Pamela Soltis15,
  • Dennis Stevenson20,
  • C. Neal Stewart Jr5,
  • Barbara Surek17,
  • Christina J. M. Thomsen1,
  • Juan Carlos Villarreal21,
  • Xiaolei Wu3,
  • Yong Zhang3,
  • Michael K. Deyholos2,
  • Gane Ka-Shu Wong2,3,22
  • 1Department of Biology‚ University of Toronto at Mississauga‚ Mississauga‚ Ontario‚ Canada;
  • 2Department of Biological Sciences‚ University of Alberta‚ Edmonton‚ Alberta‚ Canada;
  • 3BGI-Shenzhen‚ Bei Shan Industrial Zone‚ Yantian District‚ Shenzhen‚ China;
  • 4International Rice Research Institute‚ Metro Manila‚ Philippines;
  • 5Department of Plant Sciences‚ University of Tennessee‚ Knoxville‚ Tennessee‚ United States of America;
  • 6Department of Plant Biology‚ University of Georgia‚ Athens‚ Georgia‚ United States of America;
  • 7Jodrell Laboratory‚ Royal Botanic Gardens‚ Kew‚ Richmond‚ Surrey‚ United Kingdom;
  • 8Genome Institute of Singapore‚ Singapore‚ Singapore;
  • 9Department of Plant Sciences‚ University of Cambridge‚ Cambridge‚ United Kingdom;
  • 10Department of Biology and Intercollege Graduate Program in Plant Biology‚ Huck Institutes of the Life Sciences‚ Pennsylvania State University‚ University Park‚ Pennsylvania‚ United States of America;
  • 11Division of Biological Sciences‚ University of Missouri‚ Columbia‚ Missouri‚ United States of America;
  • 12Department of Botany and UBC Botanical Garden‚ University of British Columbia‚ Vancouver‚ British Columbia‚ Canada;
  • 13Max Planck Institute for Molecular Plant Physiology‚ Wissenschaftspark Golm‚ Am Mühlenberg 1‚ Potsdam-Golm‚ Germany;
  • 14Department of Biology‚ University of Florida‚ Gainesville‚ Florida‚ United States of America;
  • 15Florida Museum of Natural History‚ University of Florida‚ Gainesville‚ Florida‚ United States of America;
  • 16Donald Danforth Plant Science Center‚ St. Louis‚ Missouri‚ United States of America;
  • 17Department of Botany‚ Cologne Biocenter‚ University of Cologne‚ Cologne‚ Germany;
  • 18Department of Plant Biology‚ North Carolina State University‚ Raleigh‚ North Carolina‚ United States of America;
  • 19Department of Ecology and Evolutionary Biology‚ University of Toronto‚ Toronto‚ Ontario‚ Canada;
  • 20New York Botanical Garden‚ Bronx‚ New York‚ United States of America;
  • 21Department of Ecology and Evolutionary Biology‚ University of Connecticut‚ Storrs‚ Connecticut‚ United States of America;
  • 22Department of Medicine‚ University of Alberta‚ Edmonton‚ Alberta‚ Canada
Eric Carpenter
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.4rfgv3n
Protocol Citation: 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 2019. RNA Isolation from Plant Tissue Protocol 6: pBIOZOL and Qiagen RNeasy Plant Mini Kit Method. protocols.io https://dx.doi.org/10.17504/protocols.io.4rfgv3n
Manuscript citation:
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. https://doi.org/10.1371/journal.pone.0050226
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
These protocols were used for RNA extraction from plant tissues in order to support the One Thousand Plants initiative's work to produce RNA-Seq transcriptomes from a diverse collection of plant samples.
Created: June 26, 2019
Last Modified: August 20, 2019
Protocol Integer ID: 25095
Keywords: RNA, RNA isolation, RNA extraction, plant tissue
Abstract
This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6) 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. https://doi.org/10.1371/journal.pone.0050226
Attachments
Icon for journal.pone.0050226.s011.PDF
journal.pone.0050226...
283KB
Materials
MATERIALS
ReagentRNeasy Plant Mini KitQiagenCatalog #74904
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Grind tissue to a powder in liquid nitrogen.
AddAmount1.3 mL of cold (Temperature4 °C ) pBIOZOL reagent for up to Amount100 mg of frozen ground tissue.


Mix by briefly vortexing or flicking the bottom of the tube until the sample is thoroughly re-suspended.
Incubate the tube for Duration00:05:00 at TemperatureRoom temperature .

Note
Lay the tube down horizontally to maximize surface area during RNA extraction.



Centrifuge for Duration00:10:00 at Centrifigation12000 x g in a microcentrifuge at TemperatureRoom temperature .



Transfer the supernatant to a new Amount1.5 mL RNase-free tube.

Add Amount100 µL of Concentration5 Molarity (M) NaCl and Amount300 µL chloroform.



Vortex vigorously.
Centrifuge at Centrifigation12000 x g for Duration00:10:00 .


Transfer the top aqueous phase to a new 1.5 ml RNase-free tube.
Add an equal volume of 5:1 acid phenol:chloroform to the tube.
Vortex the tube until the phases mix and appear cloudy.
Incubate at Temperature20 °C for Duration00:05:00 .


Centrifuge at Centrifigation12000 x g for Duration00:10:00 .


Transfer the top aqueous phase to a new 1.5 ml RNase-free tube.
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 TemperatureRoom temperature for Duration00:05:00 .


Centrifuge at Centrifigation12000 x g for Duration00:10:00 .


Transfer the top aqueous phase to a new 1.5 ml RNase-free tube.
Add 1/2 volume of 100 % ethanol.
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.
Centrifuge at Centrifigation12000 x g for Duration00:00:15 .

Note
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 mini RNA spin column, until all of the liquid in the tube(s) has been passed through the column.

Note
The nucleic acid is now bound to the silica membrane in the spin column.

Apply Amount700 µL of solution RW1 to the spin column.

Cap the tube.

Centrifuge at Centrifigation12000 x g for Duration00:00:15 .
Note
The column should be empty at the end of this spin.



Discard the flow-through from the collection tube.
Apply Amount500 µL of solution RPE to the spin column.

Cap the tube
Centrifuge at Centrifigation12000 x g for Duration00:00:15 .
Note
The column should be empty at the end of this spin.



Discard the flow-through.
Repeat previous two steps one time.Go to

Spin at maximum speed for Duration00:02:00 to remove remaining liquid from the silica membrane.

Transfer the spin column to a new 1.5 ml conical bottom microcentrifuge tube.
Add Amount30 µL –Amount50 µL of RNase-free water to the column.


Then let tube incubate at Temperature20 °C for Duration00:03:00 .


Spin at maximum speed for Duration00:01:00 to collect RNA solution.