This method is derived from a modification of the protocol developed by Chirgwin et al. (1979). The reagents specified for Chirgwin et al. are commonly used in many kits and are commonly used for isolation and purification of RNA. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Preparation of RNA by ultracentrifugation with guanidine isothiocyanate/cesium chloride
Materials and Instruments
Cells and tissues Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform Cesium chloride solution Dithiothreitol EDTA Ethanol Guanidine isothiocyanate lysis buffer Isopropyl alcohol N-lauroyl sarcosine Lithium chloride Liquid nitrogen β-mercaptoethanol Phenol
Centrifuges and rotors Mortar and pestle Ultracentrifuge tubes
1. Buffers, solutions and reagents
Chloroform
Cesium chloride solution, 5.7 mol/L (5.7 mol/L cesium chloride, 25 mmol/L sodium acetate, pH 6.0, 1 mmol/LEDTA, pH 8.0)
Water treated with diethylpyrocarbonate (DEPC)
Dithiothreitol (DTT), 0.2 mmol/L
EDTA, 1 mmol/L, pH 8.0
Ethanol, 70% (v/v)
Guanidine isothiocyanate lysis buffer (4 mol/L guanidine isothiocyanate, 25 mmol/L sodium acetate, pH 6.0, 1 mmol/L EDTA, pH 8.0)
Isopropyl alcohol
N-lauroyl sarcosine, 20% (200 g/L)
Lithium chloride (LiCl), 8 mol/L
Liquid Nitrogen
β-mercaptoethanol,10 ml/L
Phenol, water saturated, pH 4.0
2. Centrifuge and rotor
Beckman Ti50 rotor or equivalent
3. Specialized equipment
Mortar and pestle, washed with DEPC-treated water, pre-cooled
Ultracentrifuge tubes (heterogeneous homocrystalline copolymer), or equivalent Polygon, or equivalent high speed homogenizer
4. Cells and tissues
Cells/tissues, frozen in liquid nitrogen
II. Methods
1. Grind samples to a fine powder under liquid nitrogen using a mortar and pestle. Homogenize tissues or cells (up to 1X109 ) in 7 ml of guanidine lysis buffer using a Polygon homogenizer or other suitable apparatus.
2. Add 4 ml of 5.7 mol/L CsCl solution to an ultracentrifuge tube.
3. 1 ml of 5.7mol/L CsCl solution, 120ul of 10mol/L non-mercaptoethanol, and 240ul of N-lauroyl sarcosine are added to the lysis products.
4. Mix the cesium chloride/cleavage product mixture, carefully layering it on top of the cesium chloride solution in the centrifuge tube.
5. Centrifuge at 180,000 g for 21 h at 20°C.
6. Remove the cesium chloride supernatant, being careful not to disturb the RNA sediment at the bottom of the tube.
7. Carefully redissolve the RNA precipitate in 750ul of guanidine isothiocyanate lysis buffer.
8. Add an equal volume of phenol and mix well.
9. Centrifuge at 10000g for 15 min.
10. Transfer the aqueous phase to a new tube. Repeat the phenol extraction of the aqueous phase until the interface is clear.
11. Add 1x volume of chloroform to the clear aqueous phase.
12. Mix and centrifuge at 10,000 g for l0 min.
13. Transfer the aqueous phase to a clean test tube. Add equal volumes of isopropyl yeast and 1/10 volume of 8 mol/L lithium chloride. 10,000 g. Centrifuge for 10 min.
14. Discard the supernatant. Wash the RNA sediment with 750ul of 70% ethanol and centrifuge at 10,000g.
15. Remove the ethanol, air-dry the sediment, and re-dissolve in 50-500ul of 0.2 mmol/L DTT.
16. Store the RNA at -70°C.