RNA Extraction Using TRIzol Reagent

Material Required:

• Chloroform
• Isopropyl alcohol
• 75% ethanol (in DEPC-treated water)
• RNase-free water or 0.5% SDS
• Centrifuge and rotor capable of reaching up to 12,000 × g
• Polypropylene microcentrifuge tubes
• Water bath or heat block (55–60°C)

Method:

Homogenizing samples
1. Determine your sample type, and perform homogenization at room temperature according to the table
below. The sample volume should not exceed 10% of the volume of TRIzol® Reagent used for
homogenization. Be sure to use the indicated amount of TRIzol® Reagent, because an insufficient
volume can result in DNA contamination of isolated RNA.
Tissues
1. Add 1 mL TRIzol® Reagent per 50–100 mg of tissue sample.
2. Homogenize sample using a glass-Teflon® or power homogenizer.
Note: Process or freeze tissue samples immediately upon collection.

Adherent Cells (Monolayer)

1. Remove growth media from culture dish.
2. Add 1 mL TRIzol® Reagent directly to the cells in the culture dish per 10 cm2 of
culture dish surface area.
Note: Add 1 mL TRIzol® Reagent for a 35 mm dish, 3 mL for a 60 mm dish, and 8 mL for a
100 mm dish.
3. Lyse the cells directly in the culture dish by pipetting the cells up and down several
times.

Suspension Cells
1. Harvest cells by centrifugation and remove media.
2. Add 0.75 mL of TRIzol® Reagent per 0.25 mL of sample (5–10 × 106 cells from
animal, plant or yeast origin, or 1 × 107 cells of bacterial origin).
Note: Do not wash cells before addition of TRIzol® Reagent to avoid increased chance of
mRNA degradation.
3. Lyse cells in sample by pipetting up and down several times. Disruption of some yeast
and bacterial cells may require the use of a homogenizer.

2. (Optional) When preparing samples with high content of fat, proteins, polysaccharides, or extracellular
material (e.g., muscle, fat tissue, or tuberous plant material), an additional isolation step may be required
to remove insoluble material from the samples.

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 Note: Do not perform this additional isolation step if you are performing subsequent DNA isolation on your
sample.

Tissue or cells with high content of fat, protein, polysaccharide, or extracellular material
1. Following homogenization, centrifuge your sample at 12,000 × g for 10 minutes at 4°C.
2. Note: The resulting pellet contains ECM, polysaccharides, and high molecular weight
DNA, while the supernatant contains the RNA. In high fat content samples, a layer of fat
collects above the supernatant.
3. Remove and discard the fatty layer.
4. Transfer the cleared supernatant to a new tube.

3. Proceed to Phase separation, or store the homogenized sample. Homogenized samples can be stored at
room temperature for several hours, or at –60 to –70°C for at least one month.

Phase separation

1. Incubate the homogenized sample (see Homogenizing samples) for 5 minutes at room temperature to
permit complete dissociation of the nucleoprotein complex.
2. Add 0.2 mL of chloroform per 1 mL of TRIzol® Reagent used for homogenization. Cap the tube
securely.
3. Shake tube vigorously by hand for 15 seconds.
4. Incubate for 2–3 minutes at room temperature.
5. Centrifuge the sample at 12,000 × g for 15 minutes at 4°C.
6. Note: The mixture separates into a lower red phenol-chloroform phase, an interphase, and a colorless
upper aqueous phase. RNA remains exclusively in the aqueous phase. The upper aqueous phase is ~50%
of the total volume.
7. Remove the aqueous phase of the sample by angling the tube at 45° and pipetting the solution out.
Avoid drawing any of the interphase or organic layer into the pipette when removing the aqueous phase.
8. Place the aqueous phase into a new tube and proceed to the RNA Isolation Procedure.
9. Save the interphase and organic phenol-chloroform phase if isolation of DNA or protein is desired. See
DNA Isolation Procedure and Protein Isolation Procedure for details. The organic phase can be
stored at 4°C overnight.

RNA Isolation Procedure

• RNA precipitation
1. (Optional) When precipitating RNA from small sample quantities (<106 cells or <10 mg tissue), add 5–
10 μg of RNase-free glycogen as a carrier to the aqueous phase.
Note: Glycogen is co-precipitated with the RNA, but does not inhibit first-strand synthesis at
concentrations ≤4 mg/mL, and does not inhibit PCR.

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 2. Add 0.5 mL of 100% isopropanol to the aqueous phase, per 1 mL of TRIzol® Reagent used for
homogenization.
3. Incubate at room temperature for 10 minutes.
4. Centrifuge at 12,000 × g for 10 minutes at 4°C.
Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and
bottom of the tube.
5. Proceed to RNA wash.

• RNA wash
1. Remove the supernatant from the tube, leaving only the RNA pellet.
2. Wash the pellet, with 1 mL of 75% ethanol per 1 mL of TRIzol® Reagent used in the initial
homogenization.
Note: The RNA can be stored in 75% ethanol at least 1 year at –20°C, or at least 1 week at 4°C.
3. Vortex the sample briefly, then centrifuge the tube at 7500 × g for 5 minutes at 4°C. Discard the wash.
4. Vacuum or air dry the RNA pellet for 5–10 minutes. Do not dry the pellet by vacuum centrifuge.
Note: Do not allow the RNA to dry completely, because the pellet can lose solubility. Partially dissolved
RNA samples have an A260/280 ratio <1.6.
5. Proceed to RNA resuspension.
6. RNA resuspension
7. Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution (20–50 μL) by passing the
solution up and down several times through a pipette tip.
Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions.
8. Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.
9. Proceed to downstream application, or store at –70°C.

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 Flow Chart
For Adherent Cells (Monolayer)

Remove growth media from culture dish.

Add 1 mL TRIzol® Reagent in the culture dish per 10 cm2 of culture dish surface area.

Lyse the cells directly in the culture dish by pipetting the cells up and down several times.

For Tissues

Add 1 mL TRIzol® Reagent per 50–100 mg of tissue sample.

Homogenize sample using a glass-Teflon® or power homogenizer.

Note: Process or freeze tissue samples immediately upon collection.

For Suspension Cells

Harvest cells by centrifugation and remove media.

Add 0.75 mL of TRIzol® Reagent per 0.25 mL of sample (5–10 × 106 cells from animal, plant or yeast origin,
or 1 × 107 cells of bacterial origin).

Lyse cells in sample by pipetting up and down several times. Disruption of some yeast and bacterial cells may
require the use of a homogenizer.

An additional isolation step may be required to remove high content of fat, proteins, polysaccharides, or
extracellular material

Following homogenization, centrifuge your sample at 12,000 × g for 10 minutes at 4°C.

Note: The resulting pellet contains ECM, polysaccharides, and high molecular weight DNA, while the
supernatant contains the RNA. In high fat content samples, a layer of fat collects above the supernatant.

Remove and discard the fatty layer.

Transfer the cleared supernatant to a new tube.

Homogenized samples can be stored at room temperature for several hours, or at –60 to –70°C for at least one
month.

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 RNA EXTRACTION THROUGH TRIZOL

Incubate the homogenized sample for 5 minutes at room temperature

Add 0.2 mL of chloroform per 1 mL of TRIzol® Reagent used for homogenization. Cap the tube securely.

Shake tube vigorously by hand for 15 seconds or Vortex

Incubate for 2–3 minutes at room temperature. Can be exceeded till 45 min RT

Centrifuge the sample at 12,000 × g for 15 minutes at 4°C.

Remove the aqueous phase of the sample.

Place the aqueous phase into a new tube. Save the interphase and organic phenol-chloroform phase if
isolation of DNA or protein is desired.

Add 0.5 mL of 100% isopropanol to the aqueous phase, per 1 mL of TRIzol® Reagent used for
homogenization.

Incubate at room temperature for 10 minutes.

Incubate at RT for 45 min at shaking. (RNA can be left at -200 C O/N)

Centrifuge at 12,000 × g for 10 minutes at 4°C.

Remove the supernatant from the tube, leaving only the RNA pellet.

Wash the pellet, with 1 mL of 75% ethanol per 1 mL of TRIzol® Reagent used in the initial
homogenization. The RNA can be stored in 75% ethanol at least 1 year at –20°C, or at least 1 week at 4°C.

Vortex the sample briefly, then centrifuge the tube at 7500 × g for 5 minutes at 4°C. Discard the wash.

Vacuum or air dry the RNA pellet for 5–10 minutes. Do not allow the RNA to dry completely.

Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution (20–50 μL) by passing the solution up
and down several times through a pipette tip.

Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.

Proceed to downstream application, or store at –70°C.

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