---+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: r2 - 2018-11-30 - SimonDAlton