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# Total RNA Extraction Protocol using TRIzol

> Published on Vicena | [View on web](https://vicena.ai/wiki/total-rna-extraction-protocol-using-trizol-RzODuA)

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## User

What is the step-by-step protocol for total RNA extraction from mammalian cells using TRIzol, including cell lysis, phase separation, isopropanol precipitation, wash steps, and how to assess RNA quality by A260/A280?

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## Vicena

The following step-by-step protocol for total RNA extraction from mammalian cells is based on standard TRIzol (phenol-guanidinium isothiocyanate) methods and quality assessment guidelines from [ThermoFisher](https://documents.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Ftrizol_reagent.pdf) and [MilliporeSigma](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-lysis-and-extraction/tri-reagent?srsltid=AfmBOop9yXz-QVTzI1p01YPWVzpkEVaBpIkLTt5W8gZrK4qQ16s2nKBk).

### **I. Cell Lysis and Homogenization**
1.  **Sample Preparation**:
    *   **Adherent Cells**: Remove growth media. Add $1\text{ mL}$ of TRIzol per $10\text{ cm}^2$ of culture surface area (e.g., a $35\text{ mm}$ dish). Lyse cells directly on the plate by pipetting up and down.
    *   **Suspension Cells**: Pellet cells by centrifugation ($300 \times g$). Add $1\text{ mL}$ of TRIzol per $5\text{--}10 \times 10^6$ cells. 
    *   *Note*: Do not wash cells with PBS before lysis to avoid mRNA degradation.
2.  **Incubation**: Incubate the lysate for **5 minutes** at room temperature ($15\text{--}25^\circ\text{C}$) to allow complete dissociation of nucleoprotein complexes.

### **II. Phase Separation**
1.  **Chloroform Addition**: Add $0.2\text{ mL}$ of chloroform per $1\text{ mL}$ of TRIzol used.
2.  **Mixing**: Shake the tube vigorously by hand for **15 seconds**. Do not vortex.
3.  **Incubation**: Incubate at room temperature for **2–3 minutes**.
4.  **Centrifugation**: Centrifuge at **$12,000 \times g$ for 15 minutes at $4^\circ\text{C}$**.
    *   The mixture will separate into three layers: a lower red organic phase (protein), a white interphase (DNA), and a **colorless upper aqueous phase** (RNA).

### **III. RNA Precipitation**
1.  **Transfer**: Carefully transfer the aqueous phase to a new RNase-free tube. Avoid touching the interphase to prevent DNA contamination.
2.  **Precipitation**: Add $0.5\text{ mL}$ of **isopropanol** per $1\text{ mL}$ of TRIzol used.
3.  **Incubation**: Incubate at room temperature for **10 minutes**.
4.  **Centrifugation**: Centrifuge at **$12,000 \times g$ for 10 minutes at $4^\circ\text{C}$**. A small, white, gel-like RNA pellet should form at the bottom.

### **IV. RNA Wash**
1.  **Ethanol Wash**: Remove the supernatant. Add $1\text{ mL}$ of **75% ethanol** per $1\text{ mL}$ of TRIzol used.
2.  **Centrifugation**: Vortex briefly and centrifuge at **$7,500 \times g$ for 5 minutes at $4^\circ\text{C}$**.
3.  **Drying**: Remove the ethanol completely. Air-dry the pellet for **5–10 minutes**. 
    *   *Critical*: Do not over-dry the pellet (it will become difficult to resuspend) or use a vacuum centrifuge.

### **V. Solubilization**
1.  **Resuspension**: Dissolve the pellet in $20\text{--}50\text{ }\mu\text{L}$ of RNase-free water or $0.5\%\text{ SDS}$.
2.  **Incubation**: Incubate at **$55\text{--}60^\circ\text{C}$ for 10–15 minutes** to ensure the RNA is fully in solution.

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### **VI. Assessing RNA Quality (A260/A280)**
RNA purity and concentration are determined using spectrophotometry (e.g., NanoDrop).

1.  **Measurement**: Measure absorbance at $260\text{ nm}$ (nucleic acids) and $280\text{ nm}$ (proteins/phenols).
2.  **Concentration Calculation**: 
    $$ \text{RNA Concentration } (\mu\text{g/mL}) = A_{260} \times \text{dilution factor} \times 40 $$
3.  **Purity Interpretation**:
    *   **Pure RNA**: An $A_{260}/A_{280}$ ratio of **$\sim 2.0$** is considered high quality.
    *   **Low Ratio ($< 1.8$)**: Indicates contamination with proteins, phenol, or other organic compounds.
    *   **High Ratio ($> 2.1$)**: May indicate a "poor quality blank" or issues with the buffer pH, as acidic solutions can lower the ratio [ThermoFisher, 2026](https://documents.thermofisher.com/TFS-Assets/CAD/Product-Bulletins/T123-NanoDrop-Lite-Interpretation-of-Nucleic-Acid-260-280-Ratios.pdf).

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