Total RNA Extraction Protocol using TRIzol

Research will be limited to:
- Substrate: Mammalian Cells
- Method: TRIzol extraction
- Molecule: Total RNA
- Analysis: A260/A280 ratio

Strategy: The user requires a detailed, step-by-step laboratory protocol for extracting total RNA from mammalian cells using the TRIzol method, followed by specific quality assessment. While the general TRIzol procedure is standard, I need to ensure the protocol includes specific volumes for mammalian cell pellets/monolayers, precise centrifugation parameters (speed/time/temperature), and the specific interpretation of A260/A280 ratios for RNA purity. I will search for a comprehensive protocol that covers lysis, phase separation, precipitation, washing, and the final spectrophotometric analysis.
Queries:
- standard TRIzol RNA extraction protocol mammalian cells step-by-step
- RNA quality assessment A260/A280 ratio interpretation mammalian cells

🔍 Reading and summarizing 5 new websites...

1. [PDF] TRIzol Reagent User Guide (Pub.No. MAN0001271 D) - ThermoFisher
2. [PDF] RNA Extraction with Trizol for cells - Protocols.io
3. TRIzol RNA Extraction Protocol - RNA / BOC Sciences
4. TRI Reagent® Protocol - MilliporeSigma
5. [PDF] Interpretation of Nucleic Acid 260/280 Ratios - ThermoFisher

Found 5 websites. Summarized content:

[PDF] TRIzol Reagent User Guide (Pub.No. MAN0001271 D) - ThermoFisher
Here's a step-by-step protocol for total RNA extraction from mammalian cells using TRIzol, based on the provided ThermoFisher TRIzol Reagent User Guide (Pub. No. MAN0001271 D):

I. Lysis and Homogenization:

1. Cell Type & TRIzol Volume:
   • Monolayer Cells: Remove growth media from cells grown in monolayer. Add 1 mL of TRIzol Reagent per 1 × 10^5 to 1 × 10^7 cells directly to the 3.5-cm culture dish (10 cm2).
   • Suspension Cells: Collect cells by centrifugation and discard the supernatant. Add 1 mL of TRIzol Reagent per 0.25 mL of sample (5-10 x 10^6 animal, plant or yeast cells; or 1 x 10^7 bacterial cells) to the pellet. Do NOT wash cells before adding TRIzol.
   • Note: The sample volume should not exceed 25% of the volume of TRIzol Reagent used for lysis.
2. Homogenization: Pipet the lysate up and down several times to homogenize.
3. Incubation: Incubate for 5 minutes at room temperature to allow complete dissociation of nucleoprotein complexes.
   • Stopping Point: Samples can be stored at 4°C overnight or at -20°C for up to a year.

II. Phase Separation:

1. Chloroform Addition: Add 0.2 mL of chloroform per 1 mL of TRIzol Reagent used for lysis.
2. Mixing: Securely cap the tube and thoroughly mix by shaking.
3. Incubation: Incubate for 2-3 minutes at room temperature.
4. Centrifugation: Centrifuge the sample for 15 minutes at 12,000 x g at 4°C. This will separate the mixture into a lower phenol-chloroform phase, an interphase, and a colorless upper aqueous phase.
5. Aqueous Phase Transfer: Angle the tube at 45° and carefully transfer the aqueous phase (containing the RNA) to a new tube, avoiding the interphase and organic layer.

III. RNA Precipitation:

1. (Optional): If the starting sample is small (< 10^6 cells or < 10mg of tissue), add 5-10 μg of RNase-free glycogen to the aqueous phase as a carrier.
2. Isopropanol Addition: Add 0.5 mL of isopropanol per 1 mL of TRIzol Reagent used for lysis.
3. Incubation: Incubate for 10 minutes at room temperature (15-30°C).
4. Centrifugation: Centrifuge for 10 minutes at 12,000 x g at 4°C. A white gel-like pellet of total RNA should form at the bottom of the tube.
5. Supernatant Removal: Discard the supernatant using a micropipettor.

IV. RNA Wash:

1. Ethanol Resuspension: Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol Reagent used for lysis.
2. Vortex and Centrifuge Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C.
   Note: The RNA can be stored in 75% ethanol for at least 1 year at –20°C, or at least 1 week at 4°C.
3. Supernatant Removal: Discard the supernatant.
4. Drying: Vacuum or air dry the RNA pellet for 5-10 minutes. Do not over-dry the pellet, and do not dry by vacuum centrifuge.

V. RNA Solubilization:

1. Resuspension: Resuspend the pellet in 20-50 μL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting up and down. Do not use 0.5% SDS if RNA will be used in enzymatic reactions.
2. Incubation: Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.

VI. A260/A280 Quality Assessment:

1. Dilution and Measurement: Dilute the sample in RNase-free water and measure the absorbance at 260 nm and 280 nm using a spectrophotometer. NanoDrop spectrophotometers can also be used without prior dilution.
2. Calculation:
   • Calculate RNA concentration: A260 × dilution factor × 40 = μg RNA/mL
   • Calculate the A260/A280 ratio.
3. Purity Assessment: An A260/A280 ratio of approximately 2 is considered indicative of pure RNA.

Important Notes:

• Wear appropriate protective equipment (gloves, eyewear, clothing).
• Use disposable, sterile, RNase-free plasticware, pipettes, and tubes.
• Work in an RNase-free environment and use RNase decontamination solutions.
• Ensure that all materials that come into contact with TRIzol are compatible with phenol, guanidine isothiocyanate, and chloroform.
• Quick-freeze samples immediately after collection and store at –80°C or in liquid nitrogen until RNA isolation, or perform RNA isolations immediately after sample collection.

[PDF] RNA Extraction with Trizol for cells - Protocols.io
Here's a step-by-step protocol for total RNA extraction from mammalian cells using TRIzol, based on the provided document:

Materials:

• RNase-free Water
• TRIzol Reagent
• Isopropanol
• Chloroform
• 75% Ethanol
• 0.1 mM EDTA or 0.5% Sodium dodecyl sulfate solution

Safety Precautions:

• TRIzol is toxic. Use gloves, lab coat, and mask in an exhaust hood.

Before Starting:

• Clean work area with 70% alcohol. Use filtered and autoclaved tips. Refrigerate centrifuge to 4°C.

Protocol:

1. Lysis: Remove culture medium and add 750 μL of TRIzol to 1 x 10^5 - 1 x 10^7 cells. Homogenize with a pipette and transfer to a tube. Optional: Freeze at -80°C for up to 6 months.
2. Incubation: Incubate for 5 minutes to allow complete dissociation of nucleoprotein complexes.
3. Phase Separation: Add 200 μL of chloroform per 1 mL of TRIzol. Incubate for 2-3 minutes.
4. Centrifugation: Centrifuge at 12,000 x g at 4°C for 15 minutes. This will separate the mixture into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase.
5. RNA Transfer: Carefully transfer the aqueous (colorless) phase (containing the RNA) to a new tube, angling the tube at 45° and pipetting out the solution.
6. RNA Precipitation: Add 500 μL of isopropanol per 1 mL of Trizol used. Incubate for 10 minutes.
7. Centrifugation: Centrifuge at 12,000 x g at 4°C for 10 minutes.
8. Wash: Discard the supernatant. Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of Trizol used.
9. Centrifugation: Vortex briefly and centrifuge for 5 minutes at 7500 x g at 4°C.
10. Drying: Discard the supernatant. Vacuum or air-dry the RNA pellet for 5-10 minutes.
11. Resuspension: Resuspend the pellet in 20-50 μL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting up and down.
12. Solubilization: Incubate in a water bath or heat block set at 55-60°C for 10-15 minutes.
13. Storage: Store at -80°C until use.

A260/A280 quality assessment: The document does not describe how to do this, just the steps for total RNA extraction using Trizol.

TRIzol RNA Extraction Protocol - RNA / BOC Sciences
Here's a step-by-step protocol for total RNA extraction from mammalian cells using TRIzol, based on the provided website content:

TRIzol RNA Extraction Protocol for Mammalian Cells

1. Reagent Preparation:

• Chloroform
• Isopropanol
• 75% Ethanol (in DEPC-treated water)
• RNase-free water

2. Cell Lysis and Homogenization:

• Adherent Cells: Directly add 1 ml of TRIzol to a 3.5 cm culture dish. Pipette to break up the lysate. Adjust TRIzol volume based on dish size (1 ml/10 cm2). Increase TRIzol volume if lysate is viscous to prevent DNA contamination.
• Suspension Cells: Centrifuge and collect cells. Add 1 ml TRIzol per (5~10) × 10^6 cells. Avoid washing to prevent mRNA degradation and RNase contamination.

3. Phase Separation:

• Add 0.2 ml of chloroform per 1 ml of TRIzol used.
• Shake vigorously for 15 seconds.
• Incubate on ice for 5 minutes.
• Centrifuge at 12,000 x g for 10 minutes at room temperature.
• This separates into three layers: a red phenol-chloroform phase (bottom), an interphase, and a colorless aqueous phase (top, containing RNA). The aqueous phase is about 60% of the TRIzol volume.

4. RNA Precipitation:

• Transfer no more than 80% of the aqueous phase to a new tube.
• Add 500 μl of isopropanol per 1 ml of TRIzol used.
• Vortex to mix thoroughly.
• Incubate at room temperature for 10 minutes.
• Centrifuge at 12,000 x g for 10 minutes at 4°C. A gel-like precipitate (RNA) should be visible.

5. RNA Wash:

• Discard the supernatant.
• Wash the RNA pellet with 75% ethanol.
• Vortex to mix.
• Centrifuge at ≤7,500 x g for 5 minutes at room temperature. Use DEPC-treated water for the 75% ethanol.

6. RNA Dissolution:

• Carefully aspirate the ethanol. Briefly centrifuge to collect residual ethanol and remove with a pipette.
• Air-dry the RNA pellet at room temperature for 5-10 minutes. Do not over-dry.
• Add an appropriate amount of DEPC-treated water to dissolve the RNA.

7. RNA Re-dissolution (Optional)
* Resuspend the RNA pellet in RNase-free water or 0.5% SDS. Pipette and let stand for 10 minutes at 55-60°C for stubborn samples with A260/280 < 1.6.

• Store at -20°C after measuring concentration.

8. RNA Quantification (A260/A280):

• Dilute 2 μl of the RNA solution in 98 μl of RNase-free water.
• Use RNase-free water as a blank.
• Measure the absorbance at 260 nm and 280 nm.
• An A260/A280 ratio between 1.8 and 2.0 indicates high purity.
• Concentrations around 1000 ng/ml are more accurate.

Important Notes and Precautions:

• Wear gloves and goggles and use a fume hood when working with TRIzol.
• Homogenized samples in TRIzol can be stored at -60 to -70°C for at least a month.
• RNA precipitate in 75% ethanol can be stored at 2-8°C for over a week or at -5 to -20°C for over a year.
  Not relevant

TRI Reagent® Protocol - MilliporeSigma
Here's a step-by-step protocol for total RNA extraction from mammalian cells using TRIzol, based on the provided MilliporeSigma website content.

Protocol: Total RNA Extraction from Mammalian Cells using TRIzol Reagent

I. Sample Preparation (Mammalian Cells)

• Monolayer Cells: Lyse cells directly on the culture dish using 1 ml of TRI Reagent per 10 cm² of the glass culture plate surface area. Pass the cell lysate several times through a pipette to form a homogenous lysate. Note: TRI Reagent is not compatible with plastic culture plates.
• Suspension Cells: Isolate cells by centrifugation. Lyse in TRI Reagent by repeated pipetting. Use 1 ml of reagent per 5-10 x 10⁶ animal cells.
• Storage: Samples homogenized or lysed in TRI Reagent can be stored at -70°C for up to 1 month.
• Optional Removal of Insoluble Material: For samples with high fat, protein, polysaccharide, or extracellular material content, centrifuge the homogenate at 12,000 x g for 10 minutes at 2-8°C after homogenization. Transfer the clear supernatant to a new tube, discarding any fatty layer.

II. Phase Separation

1. Allow the sample to stand for 5 minutes at room temperature to ensure complete dissociation of nucleoprotein complexes.
2. Add 0.1 ml of 1-bromo-3-chloropropane OR 0.2 ml of chloroform per 1 ml of TRI Reagent used.
3. Cover the sample tightly, shake vigorously for 15 seconds, and allow to stand for 2-15 minutes at room temperature.
4. Centrifuge at 12,000 x g for 15 minutes at 2-8°C. This separates the mixture into a red organic phase (protein), an interphase (DNA), and a colorless upper aqueous phase (RNA).

III. RNA Isolation

1. Transfer the aqueous phase to a fresh tube.
2. Add 0.5 ml of 2-propanol per 1 ml of TRI Reagent used in the Sample Preparation step. Mix and allow to stand for 5-10 minutes at room temperature.
3. Centrifuge at 12,000 x g for 10 minutes at 2-8°C. The RNA will form a pellet.

IV. RNA Wash

1. Remove the supernatant.
2. Wash the RNA pellet with a minimum of 1 ml of 75% ethanol per 1 ml of TRI Reagent used in the Sample Preparation step.
3. Vortex and centrifuge at 7,500 x g for 5 minutes at 2-8°C. If the RNA pellet floats, perform the wash in 75% ethanol at 12,000 x g.
4. Samples can be stored in ethanol at 2-8°C for at least 1 week and up to 1 year at -20°C.

V. RNA Solubilization

1. Briefly dry the RNA pellet for 5-10 minutes by air-drying or under vacuum. Do not completely dry the pellet.
2. Add an appropriate volume of formamide, water, or 0.5% SDS solution to the RNA pellet.
3. To facilitate dissolution, mix by repeated pipetting with a micropipette at 55-60°C for 10-15 minutes.

VI. Quality Assessment (A260/A280 Ratio)

• Final RNA preparation should be free of DNA and proteins.
• The A260/A280 ratio should be ≥1.7. A ratio <1.65 may indicate issues such as using too small a sample, not allowing the sample to stand for 5 minutes after homogenization, contamination of the aqueous phase with the phenol phase, or the final RNA pellet not being completely dissolved.

VII. Troubleshooting

• The website provides troubleshooting steps and possible causes for; low yield, low A260/280 ratio, RNA degradation, and DNA contamination.

[PDF] Interpretation of Nucleic Acid 260/280 Ratios - ThermoFisher
Here's a step-by-step protocol for total RNA extraction from mammalian cells using TRIzol, followed by quality assessment using A260/A280 ratio, based on your request and the provided ThermoFisher NanoDrop document. Remember to always follow safe laboratory practices and reagent guidelines.

Total RNA Extraction from Mammalian Cells using TRIzol (Protocol Summary)

This is a general summary; always consult the TRIzol reagent's specific instructions for the most accurate and up-to-date protocol.

1. Lysis:

   • Lyse mammalian cells directly in their culture vessel or centrifuge cells and resuspend in TRIzol reagent. Important: Use the appropriate volume of TRIzol reagent per cell number, as specified by the manufacturer (e.g., typically 1 mL TRIzol per 5-10 x 10^6 cells).

2. Phase Separation:

   • Incubate the lysate at room temperature for 5 minutes.
   • Add chloroform (typically 0.2 mL chloroform per 1 mL TRIzol used).
   • Shake vigorously for 15 seconds.
   • Incubate at room temperature for 2-3 minutes.
   • Centrifuge at 12,000 x g for 15 minutes at 4°C. This separates the mixture into an aqueous phase (containing RNA), an interphase, and an organic phase (containing DNA and proteins).

3. RNA Precipitation:

   • Carefully transfer the aqueous phase (top layer) to a new RNase-free tube.
   • Add isopropanol (typically 0.5 mL isopropanol per 1 mL TRIzol used for the initial lysis).
   • Incubate at room temperature for 10 minutes.
   • Centrifuge at 12,000 x g for 10 minutes at 4°C. A white RNA pellet should form.

4. RNA Wash:

   • Remove the isopropanol supernatant.
   • Wash the RNA pellet with 75% ethanol (typically 1 mL of 75% ethanol per 1 mL TRIzol used for the initial lysis).
   • Vortex briefly and centrifuge at 7,500 x g for 5 minutes at 4°C.
   • Carefully remove the 75% ethanol.
   • Allow the pellet to air-dry for 5-10 minutes (do not over-dry).

5. RNA Resuspension:

   • Resuspend the RNA pellet in RNase-free water or a suitable buffer (e.g., TE buffer).

A260/A280 Quality Assessment

1. Measurement: Use a spectrophotometer (e.g. NanoDrop) to measure the absorbance of the RNA sample at 260 nm and 280 nm. Follow the spectrophotometer's instructions for use, blanking, and sample preparation.
2. Ratio Interpretation:
   • A260/A280 ratio of ~2.0 is generally accepted as "pure" for RNA.
   • Low A260/A280 Ratio (<2.0): May indicate contamination with protein, phenol, guanidine, or other reagents used during extraction. Review a full spectral profile to identify contaminants using equipment such as the Thermo Scientific NanoDrop 2000 Spectrophotometer.
   • High A260/A280 Ratio (>2.0): A very high ratio can suggest a poor quality blank eliminating too much signal near the 280 nm wavelength. Review the spectral profile if the purity ratio is significantly higher than expected.
3. Important Considerations:
   • Accuracy at low concentrations: Inaccurate ratios may occur at very low nucleic acid concentrations (<10 ng/µL).
   • pH Effects: Changes in pH can affect the A260/A280 ratio. Acidic solutions can underestimate the ratio, while basic solutions can overestimate it.
   • Spectrophotometer Differences: Different spectrophotometers may yield slightly different ratios due to variations in wavelength accuracy.
4. Functionality: The best indicator of DNA or RNA quality is functionality in the downstream application of interest.