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