---+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
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Topic revision: r1 - 2017-01-23 - SimonDAlton