RNA extraction and qPCR

Phenol-chloroform RNA Extraction

Preparatory steps:

1. Make up 75% ethanol (e.g. 37.5mls ethanol + 12.5mls RNA free water)- PUT IN -20 freezer for later
2. Collect Chloroform from toxic cabinet and isopropyl alcohol(2-propanol) from alcohol cabinet and put in fume hood
3. Turn on centrifuge to ensure chilled to 4 degrees
4. Spray down workspace thoroughly with Anti-RNase spray (RNase AWAY™ Decontamination Reagent, ThermoFisher scientific) - including pipettes and gloves.
5. Retrieve tissue from -80 freezer, store on ice.

Experimental steps (in fume hood)

1. Thaw sample, either cells in TRIZOL frozen previously or for tissue, add 1ml TRIZOL and homegenise, 
2. Add 200ul of chloroform per 1ml of trizol to each sample 
3. Shake and mix for 15 seconds to mix vigorously – important to ensure sample phase separate correctly
4. Centrifuge at 12000xg for 15 minutes at 4°C 
5. Transfer aqueous phase (upper, clear liquid) to fresh 1.5 ml eppendorf tubes. Avoid transferring any Trizol (pink liquid). Appropriately dispose of waste in chlorinated waste.
6. Add 0.5ml of isopropyl alcohol to each sample, flick to mix and allow to sit on bench for 10 minutes at room temperature. During this time add 1uL of Glycoblue if necessary i.e. small amount of cells.
7.  Centrifuge at 12000xg for 10 minutes at 4°C (ensure hinge up so pellet forms consistently in a predictable location)
8. Drain contents of tube but tipping liquid into waste by hand.
9. Add 1ml of iced 75% ethanol (per 1 ml Trizol) to each sample
10. Return to centrifuge at 7500 x g for 5 minutes at 4°C 
11. Tip out ethanol and allow to dry upturned on tissue (10 minutes or so, do not over dry sample)
12. Use pipette to “hoover up” any residual droplets
13. Resuspend pellet in 20 μl of RNase free water.

Synthesis of cDNA 

Keep RNA on ice as much as possible

1. Determine the RNA concentration. We use the NanoDrop 1000 Spectrophotometer. 
2. Initialise with 1.5μl of RNA-Free h20, and blank the machine with a second sample of RNA free H20
3. Record concentrations of RNA (in ng/μl) and 260/280 ratio (which should be >1), wiping down contact points gently between samples

1. Pipette 10ul of RNA into newly labelled Eppendorf tubes, keeping all RNA on ice during the experiment.
2. Add RNA free water to the new eppenforfs. Volume required varies depending on RNA concentration. We require 1ug of RNA in each tube with a volume of 12 uL, this equates to a concentration of 83ng/ml. Thus, for a concentration of RNA “N” the formula for added RNA free water “W” is: (N/83 x10) - 10 = W
3. Vortex for 1 second
4. Retrieve the RT kit from -20°C storage (Qiagen: QuantiTect Reverse Transcription Kit). Keep the RT enzyme on ice.
5. Turn on the thermocycler.
6. Add 12uL of the RNA at 83ng/ml to labelled PCR tubes (Fisherbrand, Cat No 14230200)
7. Use control tube containing 12uL RNA free water only (to ensure the RNA-free water is not contaminated)
8. Briefly incubate the gDNA wipe-out and vortex to dissolve any precipitates
9. Add 2μl of gDNA Wipeout to each PCR tube (ensure lids are closed as much as possible)
10. Incubate at 42°C for 2 minutes and return to ice as quickly as possible.
11. Prepare the RT master mix on store on ice, the required ratio per RNA sample: RT: RT Buffer: Primer Mix is 1:4:1
12. Each PCR tube receives 6ul of master mix giving a total volume per tube of 20ul 
13. Pulse down to bottom of well on centrifuge 
14. Incubate for 15 minutes at 42°C
15. Incubate for 3 minutes at 95°C to inactivate the Reverse Transcriptase.
16. Dilute 1:5 by adding 80μl of RNase free water to each tube. 

qPCR

Reaction mixtures were prepared using TaqMan Gene Expression assays and the PerfeCTa® FastMix® II (Quanta; Cat: 95118-250), according to the manufacturer’s protocol (2ul cDNA, 5ul FAST MIX, 0.25ul gene expression assay, up to 10ul with RNase-Free water). Reactions were run on the LightCycler® 480 machine, using the pre-set FAM-hydrolysis template. The Gene Assays used were purchased from Thermofisher.

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