Haynes:TRIzol RNeasy

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RNA Miniprep - TRIzol/ RNeasy Spin Column Protocol
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.

MATERIALS

• RNaseZap (Life Technologies #AM9780)
• Qiagen RNeasy Mini kit (Qiagen #74104)
• TRIzol (Life Technologies #15596026)
• Chloroform

WARNING
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

1. 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.
2. 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

1. In the fume hood, add 200 μL chloroform to each sample per 1 mL TRIzol (100 μL per 500 μL TRIzol).
2. Make sure the lids are closed tightly. Mix by inverting for 20 seconds. Incubate at room temperature for 3 minutes.
3. 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.
4. Carefully remove the tubes from the centrifuge. Do not disturb the phases.
5. 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.
6. Discard the waste TRIzol in a labeled chemical waste bottle.
7. 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.

1. Clean work area and micropipettors with RNaseZap.
2. 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.
3. Add 700 μL Buffer RW1 into the column and spin for 30 seconds at top speed (≥ 8,000x g).
4. Transfer column into a new collection tube, add 500 μl Buffer RPE and spin 30 sec at ≥ 8,000x g. Discard flow-through.
5. Add another 500 μL Buffer RPE and spin for 2 minutes at top speed (≥ 8,000 xg). Discard flow-through.
6. Spin again (empty) for 1 minute at top speed (≥ 8,000 xg).
7. 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.