Barrick Lab :: RNAPlantPrep

---+ Isolation of Total RNA from Plant Material

Plant tissues can be tough (roots), impenetrably waxy, or contain large amounts of RNase activity (leaves). Flash freezing in liquid nitrogen then smashing them up gets around a lot of these issues. 

---+++ Materials and Reagents

Use filter tips. Keep all solutions, reagents and plastics to be used for RNA work separate from general supplies. Solutions need to be certified RNase free.

Equipment:
   * Bench top centrifuge.
   * ZYMO ZR Plant RNA MininPrep Cat # R2024 - add ethanol to buffers prior to use. Read through instructions - it differs from other column based methodologies in small but important ways (note the preparation of the final column, for example).
   * Liquid nitrgoen dewar, half filled.Liquid nitrogen
   * Two ice buckets of dry ice
   * Pestle and mortar
   * Forceps, large
   * Forceps, small
   * Metal spatula
   * Aluminium foil
   * 5ml eppendorf tube (one per tissue sample)
   * Microcentrifuge tubes (4 per sample)
   * Disruptor genie

Solutions:
   * RNase free water

For quality check:
   * Tapestation access/training and a reservation to use it - if you are not trained, you're not allowed to make a booking.
   * Tapestation RNA screentape (Agilent cat #5067-5576)
   * Tapestation RNA screentape sample buffer (Agilent cat #5067-5577)
   * Qubit RNA broadrange assay kit (Thermofiosher cat# Q10210)
---+++ Protocol

   * In one of the two buckets, create a small indent in the dry ice and place the pestle in it to cool. Lay a piece of aluminum foil on the dry ice and place the mortar on it.
   * Prepare tubes. Label with sample, date. You will need: 
      * One 5ml &lsquo;microcentrifuge&rsquo; tube per sample, chilled on dry ice.
      * One 1.5ml microcentrifuge tubes, filled with the bashing beads from one tube from the Zymo kit and on dry ice.
      * All tubes required in the Zymo kit manual from step 4 onwards.
   * Cut two 5&rsquo;&rsquo; x 5&rsquo;&rsquo; pieces of aluminium foil per sample, and leave them on dry ice to chill.
   * Once cold, place one piece of foil on the pestle and use the pestle to fit it against the walls.
   * Ensure that buffers in the Zymo kit are prepared.
   * Using a razor blade and the large forceps, cut away the leaf (or other tissue) and immediately snap freeze in liquid nitrogen.
   * WORK QUICKLY: Place the frozen leaf on the foil-covered pestle. Immediately place a second piece of foil over the leaf and use the mortar to smash up the leaf into small fragments. Remove the top foil and use the spatula to help move the leaf fragments into the appropriate 5ml tube. Return 5ml tube to dry ice.
   * Clean the spatula with 100% ethanol. Trash the foil in the biohazard.
   * Repeat steps 4-7 for remaining samples.
   * Transfer a portion (approx. 100-200mg) of the ground leaf to the pre-chilled microcentrifuge tube containing the bashing beads. Place on Disruptor for 15 seconds.
   * Immediately add 800ul of Zymo RNA lysis buffer and vortex for 15 seconds.
   * Repeat steps 10-11 for remaining samples.
   * Follow protocol in Zymo manual from step 4 to end.
   * Qubit/Tapestation Q.C.
   * Clean spatulas, forceps, pestle, mortar etc; soak in 100% ethanol with 1% SDS for two hours. Rinse in RNase free H2O. Wrap individual in foil and autoclave. <!--EndFragment-->

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Topic revision: r1 - 2017-08-15 - SimonDAlton