TITLE: RNA Isolation from Plant Tissue Protocol 2: McKenzie et al’s Qiagen hybrid method
AUTHORS: Donald J. MacKenzie, Morven A. McLean, Srima Mukerji, Margaret Green

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Jim Leebens-Mack and Charlotte Carrigan</div><div class = "text-block"><span>This protocol was developed by McKenzie et al.</span><span style = "vertical-align:super;">2</span><span> to facilitate the isolation of RNA from woody plants rich in phenolics and polysaccharides, such as grapes (Vitaceae) and fruit bearing Rosaceae (apples, cherries and pears). The protocol is provided on Qiagen’s website as an alternative method to be used in combination with their RNeasy Plant Minikit. We repeat the protocol here in the event that this protocol is removed from Qiagen’s website or readers find it difficult to obtain the original publication.</span></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 class = "text-block"><span style = "vertical-align:super;">2 </span><span>McKenzie, D.J., McLean, M.A., Mukerji, S. & Green, M. Improved RNA extraction from woody plants for the detection of         viral pathogens by reverse transcription‐polymerase chain reaction. Plant Disease 81, 222‐226 (1997).</span></div></div>

[STEPS]
?. Grind sample (up to ) in liquid nitrogen to a fine powder using a mortar and pestle.
50 mg
Incomplete grinding of the starting material will lead to reduced RNA yields.
?. Add  lysis buffer to a maximum of  of tissue powder. Vortex vigorously.
600 µl
50 mg
?. Add  of 20% sarkosyl.
60 µl
For samples with a high starch content, incubation at elevated temperatures shouldbe omitted to prevent swelling of the starting material.
?. Pipet the lysate directly onto a QIAshredder Spin Column (lilac-colored column, supplied in the RNeasy Plant Mini Kit)  placed in a 2 ml collection tube.
?. Carefully transfer the supernatant of the flow-through fraction to a new microcentrifuge tube without disturbing the cell-debris pellet in the collection tube.
It may be necessary to cut the end off the pipet tip in order to pipet the lysate onto the QIAshredder Spin Column. Centrifugation through the QIAshredder Spin Column removes cell debris and simultaneously homogenizes the lysate. While most of the celldebris is retained on the QIAshredder Spin Column, a very small amount of cell debris will pass through and form a pellet in the collection tube. Be careful not to disturb this pellet while transferring the lysate to a new microcentrifuge tube.
?. Add 0.5 volumes (usually ) ethanol (96–100%), and mix well by pipetting.
300 µl
If some lysate is lost during homogenization (step 4), reduce volume of ethanol proportionally. A precipitate may form after the addition of ethanol, but this will not affect the RNeasy procedure.
?. Continue with step 6 of the RNeasy Mini Protocol for Isolation of Total RNA from Plant Cells and Tissues and Filamentous Fungi in the RNeasy Mini Handbook.
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?. Transfer the tissue powder and liquid nitrogen to an appropriately sized tube, and allow the liquid nitrogen to evaporate. Do not allow the sample to thaw. Continue immediately with step 2.
?. Incubate at  in a water bath or heating block for  with vigorous shaking or intermittent vortexing.
70 °C
?. Centrifuge for  at maximum speed (> [rcf]).