Simultaneous Preparation of DNA, RNA and Proteins from Cells and Tissues in a One-Step Process

Summary

Like the rapid RNA extraction method reported by Chomczynski and Sacchi in 1987, this method consists of lysing cells with a single-phase solution containing guanidine isothiocyanate and phenol. The addition of chloroform produces a second (organic) phase in which DNA and proteins are extracted, leaving the RNA in the upper aqueous phase. This experiment was derived from "Molecular Cloning Laboratory Guide, Third Edition", translated by Huang Peitang et al.

Operation method

Simultaneous preparation of DNA, RNA and proteins from cells and tissues by a one-step method

Principle

Like the rapid RNA extraction method reported by Chomczynski and Sacchi in 1987, this method consists of lysing cells with a single-phase solution containing guanidine isothiocyanate and phenol. The addition of chloroform produces a second (organic) phase in which DNA and proteins are extracted and RNA is left in the upper aqueous phase.

Materials and Instruments

Source Cell or Tissue
Chloroform Ethanol Isopropanol Liquid Nitrogen Monophasic Lysis Reagents Phosphate Buffer Salt Solution RNA Precipitation Solution Sodium Acetate
Sorvall H1000 turntable or equivalent Sorvall SS-34 turntable or equivalent Colorimetric cups Homogenizers Mortar and pestle Polyethylene tube with screw cap Water baths

Move

I. Materials

1. Buffers and solutions

Chloroform

Ethanol

Isopropyl alcohol

liquid nitrogen

Single phase lysis reagent



Phosphate buffered salt solution (PBS), ice-cold

RNA precipitation solution (1.2 mol/L NaCl, 0.8 mol/L disodium _{citrate-15H2O} (no pH adjustment required))

Sodium acetate (3 mol/L, pH 5.2)

2. Cells and tissues

Source cell or tissue

3. Centrifuges and rotors

Sorvall H1000 turntable or equivalent

Sorvall SS-34 head or equivalent

4. Specialized equipment

Colorimetric cup for measuring absorbance at 260 nm

Homogenizers (e.g., Tekmar-Dohrmann's tissue homogenizer and Brinkmann's polytron homogenizer)

mortar and pestle

Polyethylene tubes with screw caps

Water bath, preset at 65°C

II.

1. Prepare cell or tissue samples for RNA isolation.

(1) Tissue

① Dissect the desired tissues and quick-freeze them directly in liquid nitrogen.

② Place 100 mg of frozen tissue in a mortar with liquid nitrogen and crush the tissue with a pestle and mortar. Keep adding liquid nitrogen during the grinding process to keep the tissue frozen.

(iii) Transfer the powdered tissue into a polyethylene screw cap tube containing 1 ml of ice-cold monophasic liquid.

④ Homogenize the tissue with a polymm homogenizer at high speed for 15-30 s at room temperature.

(2) Mammalian cells grown in suspension

① Collect the cells by centrifugation at 200~900 g (1000~3000 r/min for Sorvall H1000 head) for 5~10 min at room temperature in a tabletop centrifuge.

① Collect the cells by centrifugation at 200~900 g (Sorvall H1000 head: 1000~2000 r/min) for 5~10 min at room temperature. ② Aspirate the culture medium and resuspend the cell precipitate with 1~2 ml of sterile ice-cold PBS.

(iii) Collect the cells by centrifugation, aspirate the PBS, and add 1 ml of single-phase lysate per ¹⁰⁶ cells.

④ At room temperature, homogenize the cells with a homogenizer at high speed for 15~30s.

(3) Mammalian cells grown in monolayer

① Aspirate the culture medium and wash the cells once with 5~10 ml of sterile ice-cold PBS.

② Aspirate out the PBS, and lysed the cells with 1 ml of monophasic lysate per 90 mm diameter dish (0.7 ml of monophasic lysate per 60 mm diameter dish).

③ Transfer the cell lysate to a screw-cap polyethylene tube.

③ Transfer the cell lysate to a screw-cap polyethylene tube. ④ Homogenize the cell lysate with a homogenizer at high speed for 15~30s at room temperature.

2. incubate the cell homogenate at room temperature for 5 min to completely dissociate the nucleoprotein complex.

3. Add 0.2 ml of chloroform to each ml of single-phase lysate and mix the sample by vigorous shaking or using a vortex shaker.

4. Centrifuge the mixture at 12000 r/min (Sorvall SS-34 turntable 10,000 g) for 15 min at 4°C to separate the mixture into two phases, and transfer the upper aqueous phase to a new centrifuge tube.

5. Precipitate RNA from the aqueous phase: For every 1 ml of single-phase lysate, add 0.25 times the volume of isopropanol and 0.25 times the volume of RNA precipitant. Mix well and leave at room temperature for 10 min.

6. Collect the RNA precipitate by centrifugation at maximum speed for 10 min at 4°C. Wash the precipitate with 75% ethanol. Wash the precipitate twice with 75% ethanol and repeat the centrifugation. Remaining ethanol is aspirated with a disposable pipette tip. Leave the tube uncapped on the bench for a few minutes to allow the last traces of ethanol to evaporate. Do not allow the RNA precipitate to dry out.

7. Add 50 to 100 μl of DEPC-treated water. Store the RNA solution at -70°C.

Add SDS to a final concentration of 0.5% and heat to 65°C to help dissolve the RNA precipitate.

8. Estimate the concentration of RNA.

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