RnaPrep < Lab

---+Isolation of total RNA

---+++Reagents & Materials

Use filter tips, and designate all solutions, reagents and plastics as RNA-only. Keep separate from other general stocks. 

RNA extraction solution (needs to be made fresh before each use):

   * 18mM EDTA 
   * 0.025% SDS 
   * 1% 2-mercaptoethanol 
   * 95% formamide (RNA grade) 

Protocol:

   * Use freshly prepared cultures, or snap freeze cell pellets in liquid nitrogen, storing cell pellet on dry ice until ready for extraction.
   * Rapidly resuspend cell pellet in 500ul of RNA extraction solution.  Use pipette tip to dislodge 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.

RNA clean-up (using Zymo clean and concentrator -25 spin columns):

   * 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.
   * Make the following DNase I cocktail (for each sample add)

                        RNase-Free DNase I            5ul

                        Reaction Buffer                     75ul

   * 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 -800C.

Quality check RNA

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


-- Main.SimonDAlton - 23 Jan 2017
Barrick Lab > ProtocolList > RnaPrep
Topic revision: r2 - 2018-11-30 - 16:29:47 - Main.SimonDAlton