(r1) RNAPrep < Lab

---+Isolation of Total RNA

---+++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:

   * Set a heat block to 95°C.
   * A bench top centrifuge is required.
   * ZYMO RNA Clean & Concentrator™- 25 kit Cat # R1017 - add ethanol to buffers prior to use.
   * Tapestation access/training
   * Qubit

Solutions:

   * RNA extraction solution (needs to be made fresh before each use).
      * 18mM EDTA 
      * 0.025% SDS 
      * 1% 2-mercaptoethanol 
      * 95% formamide (RNA grade)
   * RNase free water
   * DNase 1 solution for on-column digestion (from Zymo, cat #E1010)
   * Ethanol, 95-100% (from Biosci store is fine)
   * Ethanol, 80% (diluted from above with RNase free water)

For quality check:

   * 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

   * Ensure DNase 1 is thawed.
   * Pellet cells and prepare immediately _OR_ flash freeze on liquid nitrogen and store at -80. When removing cells from -80 for prep, store on dry ice until ready for extraction.  
   * *Critical* _Rapidly_ resuspend cell pellet in 500ul of RNA extraction solution.  Use pipette tip to dislodge a frozen pellet, break apart and triturate until homogeneous. Work quickly to avoid RNase degradation.
   * Incubate sample at 95°C for 7min to lyse cells.  Place a weight on top of tubes (or use tube locks) to prevent pressure from opening tubes.
   * Pellet the warm sample by centrifuging at 16 000 x g for 5min at room temperature.  
   * Transfer 200ul of supernatant containing RNA to a fresh tube and continue with below RNA clean up. Store the remaining ~300ul at -80°C, as a back up.

The following uses the Zymo clean and concentrator kit:

   * Add 2 volumes (400ul) of RNA Binding Buffer to each volume of RNA sample and mix well.
   * Add 1 volume (600ul) ETOH (95-100%) to the mixture and mix well.
   * Transfer the mixture to the Zymo-Spin IIC column in a collection tube and centrifuge at >12,000 x g for 1 min.  Discard the flow through.
   * During this spin, prepare the DNase 1 cocktail for use. Per reaction:
      * 5ul RNase-Free DNase I  
      * 75ul Reaction Buffer 
   * Add 400ul of 80% ethanol to the Zymo-Spin IIC column in a collection tube and centrifuge at >12,000 x g for 30 secs.  Discard the flow through.
   * Add 80ul DNase I cocktail directly to the matrix of the Zymo-Spin IIC column.
   * Incubate the column at 25-370C for > 15 mins (optimal temp for DNase I is 370C). 
   * Centrifuge > 12,000 x g for 30 seconds. Discard the flow through.
   * Add 400ul RNA Prep Buffer to the column and centrifuge at 12,000 x g for 1 minute.  Discard the flow trough.
   * Add 800ul RNA Wash Buffer to the column and centrifuge at > 12,000 x g for 30 seconds.  Discard the flow through.  Repeat this wash step with 400ul RNA Wash Buffer.
   * Centrifuge the Zymo-Spin IIC Column in an emptied collection tube at >12,000 x g for 2 minutes.  Remove the column carefully from the collection tube and transfer to new RNase-free tube.
   * Add 30 ul of DNase/RNase-Free water directly to the column matrix and let stand for 1 minute at room temperature.
   * Centrifuge at 10,000 x g for 1 min.
   * Use the eluted RNA immediately or store at -80C.

---+++Quality check RNA

    Assess the RIN and quantity of RNA eluted using the Tapestation (load ~100ng) and Qubit (a dilution of 1/10 will be required to reach linear range), respectively.



-- Main.SimonDAlton - 23 Jan 2017
Barrick Lab > ProtocolList > RNAPrep
Topic revision: r1 - 2017-01-23 - 16:42:36 - Main.SimonDAlton