21/05/2025
Q-by-Step Guide to TRIzol RNA Extraction
1. Prepare Your Workspace
* Safety First: TRIzol contains hazardous chemicals like phenol and chloroform. Always work in a chemical fume hood and wear appropriate personal protective equipment (PPE), including gloves, lab coat, and safety goggles.
* RNase-Free Environment: Ensure all tubes, pipette tips, and water are RNase-free to prevent RNA degradation.
2. Sample Collection and Homogenization
* Tissue Samples:
* Weigh approximately 50 mg of tissue.
* Add 1 mL of TRIzol reagent per 50 mg of tissue.
* Homogenize the tissue thoroughly using a homogenizer or by passing through a syringe with a needle.
* Adherent Cells:
* For a 6-well plate, add 1 mL of TRIzol per well.
* For a 12-well plate, add 500 µL of TRIzol per well.
* Lyse the cells directly in the culture dish by adding TRIzol and pipetting up and down to ensure complete lysis.Bitesize Bio+1ZYMO RESEARCH+1
3. Incubation
* Let the homogenized samples sit at room temperature (20–25°C) for 5–10 minutes. This allows for the complete dissociation of nucleoprotein complexes.
4. Phase Separation
* Add 0.2 mL of chloroform per 1 mL of TRIzol used.
* Cap the tubes securely and shake vigorously by hand for 15 seconds.
* Incubate the samples at room temperature for 2–3 minutes.
* Centrifuge the samples at 12,000 × g for 15 minutes at 4°C.
* After centrifugation, the mixture will separate into three layers:
* Lower red phenol-chloroform phase.
* Interphase.
* Upper colorless aqueous phase (contains RNA).UConn Health+4rnaseqcore.vet.cornell.edu+4ZYMO RESEARCH+4ZYMO RESEARCH+3UConn Health+3Bitesize Bio+3ResearchGate+2Bitesize Bio+2UConn Health+2
5. RNA Precipitation
* Carefully transfer the upper aqueous phase to a new RNase-free tube without disturbing the interphase.
* Add 0.5 mL of isopropanol per 1 mL of TRIzol used initially.
* Mix the samples by inverting the tubes several times.
* Incubate at room temperature for 10 minutes.
* Centrifuge at 12,000 × g for 10 minutes at 4°C.
* The RNA will form a gel-like pellet at the bottom of the tube.ResearchGate+2Bitesize Bio+2ZYMO RESEARCH+2ResearchGateZYMO RESEARCHUConn Health
6. RNA Wash
* Remove the supernatant carefully without disturbing the RNA pellet.
* Wash the pellet with 1 mL of 75% ethanol per 1 mL of TRIzol used.
* Vortex the sample briefly and centrifuge at 7,500 × g for 5 minutes at 4°C.
* Remove the ethanol and air-dry the RNA pellet for 5–10 minutes. Avoid over-drying.
7. RNA Resuspension
* Dissolve the RNA pellet in an appropriate volume (e.g., 20–50 µL) of RNase-free water.
* Incubate at 55–60°C for 10 minutes to facilitate dissolution.
8. RNA Quantification
* Use a spectrophotometer to measure RNA concentration and purity.
* An A260/A280 ratio between 1.8 and 2.0 indicates pure RNA.
🧰 Materials and Reagents Checklist
* Reagents:
* TRIzol reagent.
* Chloroform.
* Isopropanol (100%).
* Ethanol (75%).
* RNase-free water.
* Consumables:
* RNase-free microcentrifuge tubes (1.5 mL or 2 mL).
* RNase-free pipette tips.
* Disposable gloves.
* Equipment:
* Homogenizer or syringe with needle.
* Vortex mixer.
* Refrigerated centrifuge capable of 12,000 × g.
* Spectrophotometer.
* Heat block or water bath (55–60°C).
* Chemical fume hood.
Remember, practice and consistency are key. Always follow safety guidelines and manufacturer instructions. If you need further assistance or have questions about subsequent steps like cDNA synthesis or RT-qPCR, feel free to ask!
Dr-mohamet isse Ali
