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 half 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.
• Load the remaining half and repeat spin, discard.
• 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.
• Add 400ul RNA Wash Buffer to the column and centrifuge at > 12,000 x g for 2 minutes. Discard the flow through. 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