by Karmella Haynes, 2013
Principle: Cells are lysed in TRIzol (a commercial phenol-based solution), and the RNA is collected via aqueous/ organic phase separation. Instead of using the traditional alcohol-based precipitation method to create an RNA pellet, a commercial RNA-binding column is used to purify the RNA.
- RNaseZap (Life Technologies #AM9780)
- Qiagen RNeasy Mini kit (Qiagen #74104)
- TRIzol (Life Technologies #15596026)
TRIzol is a hazardous chemical solution containing guanidinium thiocyanate, phenol, and chloroform. Exposure to TRIzol can be a serious health hazard. Always wear gloves, a lab coat, and safety goggles when handling TRIzol. Perform all steps involving TRIzol in the fume hood to prevent accidental exposure. Dispose of waste TRIzol in a labeled chemical waste bottle - do not discard in the autoclaveable waste!
LYSE CELLS IN TRIZOL
- Suspension cells: Add up to 1x10^6 cells to a 2.0 mL microfuge tube. Spin for 5 min. at 800 xg (~3000 rpm in a standard table top centrifuge) at room temp. Discard supernatant, transfer samples to the fume hood, and add 500 μL TRIzol (per 1x10^6 cells). Mix by pipetting up and down. Incubate at room temperature for 5 min. Store at -80°C or go on to the next step.
- Adherent cells: For cells grown to 100% confluency in a 6-well dish, aspirate off the growth medium, transfer samples to the fume hood, and add 500 μL TRIzol to the cells. Incubate at room temperature for 5 minutes. Using a 1000 μL pipettor w/ disposable tip, scrape the bottom of the well to detach remaining cell debris, and transfer the total solution to a 2.0 mL microfuge tube. Store at -80°C or go on to the next step.
EXTRACT RNA - PHASE SEPARATION
- In the fume hood, add 200 μL chloroform to each sample per 1 mL TRIzol (100 μL per 500 μL TRIzol).
- Make sure the lids are closed tightly. Mix by inverting for 20 seconds. Incubate at room temperature for 3 minutes.
- Centrifuge samples at 12,000 xg for 15 minutes at 4°C. The mixture will separate into a clear upper aqueous phase, a cloudy interphase, and a pink lower organic (phenol/ chloroform) phase.
- Carefully remove the tubes from the centrifuge. Do not disturb the phases.
- Bring your samples to the fume hood. Using a micropipette, carefully transfer the clear aqueous phase to a fresh RNase-free 1.5 mL tube. The aqueous phase volume should equal ~60% of the original TRIzol volume.
- Discard the waste TRIzol in a labeled chemical waste bottle.
- Slowly add an equal volume of RNase-free 70% EtOH to each sample.
SPIN-COLUMN RNA PURIFICATION
- Make sure that ethanol has been added to Buffer RPE.
- Clean work area and micropipettors with RNaseZap.
- Load up to 700 μL of sample* into an RNeasy spin column (seated in a collection tube). Spin for 30 seconds at top speed (≥ 8000 xg). Discard the flow-through. *Note: If the sample size is greater than 700, repeat this step, reusing the same column.
- Add 700 μL Buffer RW1 into the column and spin for 30 seconds at top speed (≥ 8,000x g).
- Transfer column into a new collection tube, add 500 μl Buffer RPE and spin 30 sec at ≥ 8,000x g. Discard flow-through.
- Add another 500 μL Buffer RPE and spin for 2 minutes at top speed (≥ 8,000 xg). Discard flow-through.
- Spin again (empty) for 1 minute at top speed (≥ 8,000 xg).
- Transfer column into a fresh RNase-free 1.5 mL collection tube. Pipette 30-50 μL of RNase-free water directly onto the column membrane. Allow the sample to sit at RT for 1-2 min, and then spin 1 min at top speed (≥ 8,000 xg) to elute the RNA. Store at -80°C for up to one month.