RNA Extraction with TRIzol
Introduction
Materials
- TRIzol™ Reagent
- RNase-free microcentrifuge tubes
- RNase-free pipette tips and tubes
- Isopropanol (100%)
- Ethanol (75%)
- Chloroform (molecular biolog
- Centrifuge (Refrigerated at 4ºC)
- Ice bucket
- Pipettes (P20, P200, P1000)
Procedure
I. Cell Lysis & Homogenization
- Add 1mL of TRIzol reagent directly to the cells or tissue sample.
(Pellet is typically harvested from 10 mLs of 80% confluent culture.)
- Thoroughly mix the TRIzol reagent with the cells by pipetting up and down to ensure complete lysis and homogenization.
DO NOT VORTEX RNA is a very sensitive molecule.
- Incubate the cell-TRIzol mixture at room temperature for a ~3 mins to allow the reagent to disrupt cell membranes and release RNA.
- Move mixtures to ice bucket.
- PAUSEStore lysate-TRIzol mixtures at -80ºC for use in a later date.
II. Phase Separation
- (If applicable) Thaw cell-TRIzol mixture in an ice-bucket.
- Add a fresh chloroform to the cell-TRIzol mixture in a ratio of 0.2mL per 1mL of Trizol used initially.
- Shake tubes for 15 seconds and incubate at room temperature for 3 mins.
- Centrifuge at 12,000 x g at 4ºC for 15 min.
After centrifugation, the Trizol reagent creates a biphasic mixture containing an aqueous phase (containing exclusively RNA) and an interphase and organic phase (containing DNA, proteins and other cellular components). The volume of the aqueous phase is about 60% of the volume of TRIzol Reagent used for homogenization.
- Carefully transfer the aqueous phase containing the RNA into a new microtube, taking care not to disturb the interphase or organic phase.
In this step you are separating the RNA from the rest of the cellular components. Save the organic phase if isolation of DNA or protein is desired.
RNA Precipitation:
- Precipitate the RNA from the aqueous phase by adding a 500µL of isopropanol alcohol per 1mL of TRI-zol Reagent used in the previous steps.
- Mix the solution thoroughly by gently pipetting up and down and then inverting the tube to ensure RNA precipitation.
- Incubate the mixture at -20°C for at least 15 minutes to allow the RNA to precipitate. The low temp will help the precipitation process.
- Centrifuge at 12,000 x g at 4ºC for 15 min.
A visible RNA pellet should form after centrifugation.
RNA Pellet Collection and Wash:
- Discard the supernatant carefully, without disturbing the RNA pellet.
Note: Recommended to use a pipette to carefully remove the supernatant.
- Wash the RNA pellet twice to remove residual salts and impurities.
- Add ice-cold 75% ethanol and pipette up and down, making sure to to direct the ethanol stream to the pellet and that the pellet doesn’t come up into the pipette tip. This will ensure you are washing out any salt impurities embedded in the pellet. Centrifuge at 12,000 x g at 4ºC for 15 min.
- Remove the ethanol completely using a micropipette without disturbing the RNA pellet
- Air-dry the RNA pellets (~15 minutes or more until visibly dry).
RNA Resuspension:
- Resuspend the RNA pellet in 50µL of nuclease-free water or RNase-free Tris-EDTA (TE).
- Pipette up and down the tube to ensure complete dissolution of the RNA. (DO NOT VORTEX)
- Store RNA samples at -20ºC (or -80ºC for long term storage > month).
Check RNA integrity
- Use 1-3µL of RNA to assess sample integrity using agarose bleach gel electropheresis.
Quantify RNA concentration
- Use a Nanodrop or other spectrophotometer to measure the RNA concentration.
- Record concentration and any odd-looking spectra. Use a Nanodrop to quantify RNA concentration
This protocol is for purifying RNA to prepare it for downstream applications such as reverse transcription, gene expression analysis, or RNA sequencing. TRIzol™ Reagent is a complete, ready-to-use reagent for the isolation of high-quality total RNA from biological samples. This monophasic solution of phenol and guanidine isothiocyanate is designed to isolate separate fractions of RNA, DNA, and proteins from cell and tissue samples within one hour. The protocol includes steps for precipitating and isolation the RNA fraction.
⚠ Safety Precautions:
Handel TRIzol and chlorophorm in a chemical hood. Avoid breathing vapor. Always use gloves protection. Avoid contact with skin or clothing.
Authors: Osvaldo D. Rivera, PhD
References: TBA