Extraction of RNA from cultured cells by the CsCl method
RNA preparation by guanidine thiocyanate extraction and cesium chloride centrifugation is suitable for isolation of RNA from cells with difficult separation of cytoplasm and nucleus as well as from RNase-rich cells.
Operation method
Extraction of RNA from cultured cells by the CsCl method
Materials and Instruments
Phosphate buffer solution Guanidine thiocyanate solution CsCl TES buffer Sodium acetate buffer Anhydrous ethanol DEFC-treated water. Move I. Materials and equipment Caveat 1) Allow sufficient time for re-solubilization of the precipitate, otherwise there will be a significant decrease in the yield of RNA.2) Small RNAs do not precipitate efficiently when centrifuged in CsCl, so molecules such as 5SRNA and tRNA cannot be prepared by this method.3) Ethanol can wash away cesium chloride from the precipitate. It makes the precipitate easy to dissolve For more product details, please visit Aladdin Scientific website.
Centrifuge Syringe and 20W needle
1) Phosphate buffer solution (PBS)
2) Guanidine thiocyanate solution: 4 mol/L guanidine thiocyanate, 0.lmmol/LTris-HClpH7.5, 1% β-mercaptoethanol
3) CsCl5.7mol/:L (DEPC treatment)
4)TES buffer:0.1% SDS, 10 mmol/LTris-ClpH7.4, lmmol/LEDTA
5)3 mmol/L sodium acetate buffer (PH5.2, DEPC treatment)
6) Anhydrous ethanol
7) DEFC-treated water
8) Centrifuge
9) 6 ml syringe with 20W needle
II. Operation Methods
1. Single layer cell culture
1) Wash the cells with PBS at room temperature, 5 ml per dish twice.
2) Add 3.5 ml of guanidine thiocyanate solution to no more than 105 cells to spread throughout the dish. Scrape the dish with a rubber cell scraper to recover the viscous cell lysate and aspirate the lysate back and forth with a syringe fitted with a 20-G needle, rounding it up.
2. Suspension of cultured cells
1) Centrifuge to recover ≤l08 cells, resuspend with PBS equivalent to 1/2 the original volume, and centrifuge again to recover the cells.
2) Add 3.5 ml of guanidine thiocyanate solution to the centrifuge tube.
3) The viscous cell lysate is aspirated back and forth 4 times with a 6 ml syringe fitted with a 20-G needle and transferred to a clean tube. This step is important to shear the chromosomal DNA to reduce the viscosity of the solution.
4) Add 1.5 ml of 5.7mol/LCsCl to a 13 mmX51 mm ultracentrifuge tube under high pressure in a silica well and add 3.5 ml of cell lysate to the CsCl layer, keeping the interface clear. steps 1) to 4) were performed at room temperature.
5) Centrifuge at 18℃, 150,000g for 12-20 h (slowly accelerate and decelerate).
6) Aspirate the supernatant with the tip of a Pasteur pipette at the liquid level, with the tube dropping as the liquid level drops. When only about 100 ul is left, carefully invert the tube and pour out the remaining liquid. There should be a white band of DNA at the interface, which must be carefully and completely removed because it contains chromosomal DNA.
7) Allow the precipitate to dry for about 5~lOmin, then redissolve the precipitate with 360ul of TES solution, repeatedly pipetting up and down, and so on for 5~l0 min at room temperature, and transfer the solution to another clean microcentrifuge tube.
8) Add 40u 13 mol/L pH5.2 sodium acetate buffer and 1 ml anhydrous ethanol, precipitate for 30 mim in dry ice/ethanol, centrifuge for 10-15 min, re-dissolve the precipitate in 360 ul of water and repeat the precipitation process.
9) The precipitate was allowed to dry for l0 min, dissolved in 20 ul of water, and diluted to lml with 10 ul of water to measure A260 and A280. Finally, the RNA was stored as an aqueous solution or ethanol precipitate at 70℃.