TRI Reagent® solution (also sold as TRIzol) is a mixture of a mixture of guanidine thiocyanate and phenol in a monophase solution that is used for the isolation of DNA, RNA and protein from biological samples of human, animal, plant, yeast, bacteria, and virus. It inhibits RNase activity. TRI Reagent® is used to homogenize the biological sample from which RNA, DNA or proteins are extracted.
Homogenize tissue samples in TRI Reagent (1 ml per 50–100 mg of tissue) in a Polytron® or other appropriate homogenizer.
Note: If minimal shearing of the DNA is desired, use a loosely fitting homogenizer, not a Polytron (see DNA Isolation, step 3, note b). The volume of the tissue should not exceed 10% of the volume of the TRI Reagent.
1B. Monolayer cells:
Lyse cells directly on the culture dish. Use 1 ml of the TRI Reagent per 10 cm2 of glass culture plate surface area. After addition of the reagent, the cell lysate should be passed several times through a pipette to form a homogenous lysate.
Note: TRI Reagent is not compatible with plastic culture plates.
1C. Suspension cells:
Isolate cells by centrifugation and then lyse in TRI Reagent by repeated pipetting. One ml of the reagent is sufficient to lyse 5–10 × 106 animal, plant, or yeast cells, or 107 bacterial cells.
Phase Separation: To ensure complete dissociation of nucleoprotein complexes, allow samples to stand for 5 minutes at room temperature. Add 0.1 ml of 1‑bromo-3‑chloropropane or 0.2 ml of chloroform (see Phase Separation, note a and b) per ml of TRI Reagent used. Cover the sample tightly, shake vigorously for 15 seconds, and allow to stand for 2–15 minutes at room temperature. Centrifuge the resulting mixture at 12,000 × g for 15 minutes at 2–8 °C. Centrifugation separates the mixture into 3 phases: a red organic phase (containing protein), an interphase (containing DNA), and a colorless upper aqueous phase (containing RNA).
1. Transfer the aqueous phase to a fresh tube and add 0.5 ml of 2-propanol per ml of TRI Reagent used in Sample Preparation, step 1 and mix. Allow the sample to stand for 5–10 minutes at room temperature. Centrifuge at 12,000 × g for 10 minutes at 2–8 °C. The RNA precipitate will form a pellet on the side and bottom of the tube.
Note: Store the interphase and organic phase at 2–8 °C for subsequent isolation of the DNA and proteins.
2. Remove the supernatant and wash the RNA pellet by adding a minimun of 1 ml of 75% ethanol per 1 ml of TRI Reagent used in Sample Preparation, step 1. Vortex the sample and then centrifuge at 7,500 × g for 5 minutes at 2–8 °C.
3. Briefly dry the RNA pellet for 5–10 minutes by air-drying or under a vacuum. Do not let the RNA pellet dry completely, as this will greatly decrease its solubility. Do not dry the RNA pellet by centrifugation under vacuum (Speed-Vac®). Add an appropriate volume of formamide, water, or a 0.5% SDS solution to the RNA pellet. To facilitate dissolution, mix by repeated pipetting with a micropipette at 55–60 °C for 10–15 minutes.
1. Carefully remove the remaining aqueous phase overlaying the interphase and discard. To precipitate the DNA from the interphase and organic phase, add 0.3 ml of 100% ethanol per 1 ml of TRI Reagent used in Sample Preparation, step 1. Mix by inversion and allow to stand for 2–3 minutes at room temperature. Centrifuge at 2,000 × g for 5 minutes at 2–8 °C.
Note: Removal of the remaining aqueous phase before DNA precipitation is a critical step for the quality of the isolated DNA.
2. Remove the supernatant and save at 2–8 °C for protein isolation. Wash the DNA pellet twice in 0.1 M trisodium citrate, 10% ethanol solution. Use 1 ml of wash solution for every 1 ml of TRI Reagent used in Sample Preparation, step 1. During each wash, allow the DNA pellet to stand (with occasional mixing) for at least 30 minutes. Centrifuge at 2,000 × g for 5 minutes at 2–8 °C. Resuspend the DNA pellet in 75% ethanol (1.5–2 ml for each ml TRI Reagent) and allow to stand for 10–20 minutes at room temperature.
3. Dry the DNA pellet for 5–10 minutes under a vacuum and dissolve in 8 mM NaOH with repeated slow pipetting with a micropipette. Add sufficient 8 mM NaOH for a final DNA concentration of 0.2–0.3 mg/mL (typically 0.3–0.6 ml to the DNA isolated from 50–70 mg of tissue or 107 cells). This mild alkaline solution assures complete dissolution of the DNA pellet. Centrifuge at 12,000 × g for 10 minutes to remove any insoluble material and transfer the supernatant to a new tube.
After dissolving in 8 mM NaOH, adjust to pH 8.4 using HEPES (add 86 µL of 0.1 M HEPES, free acid/ml of DNA solution). Add sample (generally 0.1–1 µg) to PCR mix and follow PCR protocol.
Adjust the pH of the DNA solution to that needed for the restriction enzyme digestion using HEPES, or dialyze samples against 1 mM EDTA, pH 7–8. Allow the restriction enzyme digestion to continue for 3–24 hours under optimal conditions. It is recommended that 3–5 units of enzyme be used per 1 µg of DNA. Typically, 80–90% of the DNA is digested.
1. Precipitate proteins (see note) from the phenol-ethanol supernatant (DNA Isolation, step 2) with 1.5 ml of 2-propanol per 1 ml of TRI Reagent used in Sample Preparation, step 1. Allow samples to stand for at least 10 minutes at room temperature. Centrifuge at 12,000 × g for 10 minutes at 2–8 °C.
Note: For some samples, the protein pellet may be difficult to dissolve in 1% SDS (step 3). Use this alternate procedure to correct the problem:
2. Discard supernatant and wash pellet 3 times in 0.3 M guanidine hydrochloride to 95% ethanol solution, using 2 ml per 1 ml of TRI Reagent used in Sample Preparation, step 1. During each wash, store samples in wash solution for 20 minutes at room temperature. Centrifuge at 7,500 × g for 5 minutes at 2–8 °C. After the 3 washes, add 2 ml of 100% ethanol and vortex the protein pellet. Allow to stand for 20 minutes at room temperature. Centrifuge at 7,500 × g for 5 minutes at 2–8 °C.
Note: Protein samples suspended in 0.3 M guanidine hydrochloride to 95% ethanol solution or 100% ethanol can be stored for 1 month at 2–8 °C or 1 year at –20 °C.
3. Dry protein pellet under a vacuum for 5–10 minutes. Dissolve pellet in 1% SDS aided by working the plunger of micropipette with tip in the solution. Remove any insoluble material by centrifugation at 10,000 × g for 10 minutes at 2–8 °C. Transfer supernatant to a new tube. The protein solution should be used immediately for Western blotting or stored at –20 °C.
1. RNA Isolation:
A. Low yield may be due to:
B. If the A260 to A280 ratio is <1.65:
C. If there is degradation of the RNA:
D. If there is DNA contamination:
2. DNA Isolation:
A. Low yield may be due to:
B. If the A260 to A280 ratio is <1.70:
C. If there is degradation of the DNA:
D. If there is RNA contamination:
3. Protein Isolation:
A. Low yield may be due to:
B. If there is degradation of the protein:
C. If PAGE shows band deformation:
I. Isolation of Poly A+ RNA
After the RNA has been precipitated with 2-propanol (RNA Isolation, step 1), dissolve the pellet in poly A+ binding buffer and pass through an oligo‑dT cellulose column to selectively remove mRNA according to the procedure of Aviv and Leder.3
II. Isolated RNA is to be used in RT-PCR
1. Modifying the procedure by performing the additional centrifugation step in the initial Sample Preparation, step 1B, note c further minimizes the possibility of DNA contamination in the RNA extracted by TRI Reagent LS.
2. A more complete evaporation of ethanol is required when RNA samples are to be used in RT-PCR. This is especially critical for small volume samples (5–20 μL), which may contain a relatively high level of ethanol if not adequately dried.
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