Youssef Farhat, Written 8/8/12, Last updated 5/6/14
Extracting high quality and quantity of RNA from cells in monolayer is essential for many gene expression experiments. This protocol was developed for fibroblasts culture in 6-well tissue culture plates.
Note that the volume of 70% EtOH to be used in step 8 of the procedure is meant to be equal to the volume of the aqueous layer obtained after the TRIzol separation (step 9). When performing this protocol for the first time, it is essential to establish the volume of clear layer that can be obtained from the TRIzol separation (step 9) and then adjust the volume of EtOH used in step 8 accordingly to equal that volume.
Several of the reagents used in this procedure are harmful to health (i.e. TRIzol and Chloroform). Wear eye, face, and hand protection as well as a lab coat throughout the procedure and be sure to review all safety information that comes with these products before using them.
- TRIzol (Invitrogen, Cat. #15596-026)
- Chloroform (Sigma-Aldrich, Cat. #C2432-25ML)
- 70% EtOH (RNase-free)
- Epoch Life Science’s GenCatch Total RNA Miniprep Kit (Cat. #1660050)
- 3 RNase-free Eppendorf tubes per sample
- RNase free water (Gibco, Cat. #10977)
- Typical lab supplies such as micropipettors and pipette tips
- Remove media from each well of the 6-well plate.
- Add 1 mL TRIzol to each well with a micropipettor and pipette up and down until the cells are completely dissolved.
- Transfer the TRIzol/cell homogenate to 1.5 mL Eppendorf tubes.
- Vortex the tubes for 5 seconds.
- Allow the samples to sit for 5 min at 20ºC.
- Add 200 μL chloroform to each tube, shake them vigorously for 15 sec, and then wait 2 min at 20ºC for the phases to separate.
- Centrifuge the tubes at 12,000 g for 15 min (5ºC) and, while waiting, pre-label 2 new RNase-free Eppendorf tubes and one Minispin column for every sample.
- Fill one of the newly labeled Eppendorf tubes with 400 µL of 70% EtOH.
- After centrifugation is complete, transfer 400 µL of the upper aqueous phase of each sample to the appropriate EtOH-filled Eppendorf tube and mix this phase thoroughly with the EtOH by pipetting several times.
- When removing the upper aqueous phase, be careful not to disturb the other layers of the TRIzol separation, as this will contaminate the RNA.
- Add 400 µL of the EtOH-RNA solution to the pre-labeled Minispin columns.
- Centrifuge at 8000 g for 15-30 sec (5ºC) & carefully empty the flow-through so as not to let any liquid touch the bottom of the column (to prevent contamination of the RNA)
- Repeat steps 10 and 11 with the remaining EtOH-RNA
- Add 500 μL WF Buffer to each column, centrifuge at 8000 g for 15 sec, & empty flow-through
- Add 700 μL WS Buffer to each column, centrifuge at 8000 g for 15 sec, empty flow-through, & discard the collection tube
- Place columns in new, unlabeled 1.5 mL Eppendorf tubes and centrifuge at 8000 g for 3 min to eliminate any remaining wash solution from the columns
- Place columns in the second set of 1.5 mL Eppendorf tubes that were labeled in step 7
- Add 34 μL RNase-free water directly to membrane and let set 1 min at 20ºC
- Centrifuge at 8000 g (5ºC) for 2 min
- The eluted water should contain RNA which is ready to be measured and reverse-transcribed. See our post on Measuring RNA with NanoDrop for more information.
- When finished, store the remaining RNA at -80ºC
Citing this Protocol