Cytoplasmic RNA extraction
Cytoplasmic RNA extraction
Since there are many sources of RNA species, there are different methods of extraction and preparation, generally phenol method, detergent method and guanidine hydrochloride method, of which phenol method is most commonly used in the laboratory. Tissue homogenization and centrifugation with phenol treatment, RNA is dissolved in the upper layer of the aqueous phase saturated with phenol, to the aqueous phase after the addition of cold ethanol, RNA that is precipitated as a white flocculent precipitate. This method is better to remove DNA and protein, and to obtain biologically active RNA.
Operation method
basic program
Materials and Instruments
RNA Move 1. For monolayer pegylated cells For more product details, please visit Aladdin Scientific website.
PBS Cell lysate SDS Proteinase K Phenol Chloroform Isoamyl alcohol Anhydrous ethanol
Centrifuge Incubator
(1) For every 10 cm dish of cultured cells, wash 3 times with ice-cold PBS.
(2) 1 ml each time Then scrape the cells with a small volume of PBS and transfer to a centrifuge tube in an ice bath.
(3) Centrifuge at 300 g for 5 min, discard the supernatant, and continue the ice bath.
2. For suspension culture cells
(1) Centrifuge at 300 g for 5 min, discard the supernatant.
(2) Resuspend the cells in 1/2 the original volume on ice, centrifuge again and discard the supernatant.3. Resuspend the cells in 375 μl of ice-cold cell lysis buffer and place in an ice bath for 5 min.
4. Centrifuge the cells in a microcentrifuge at 4°C for 2 min, transfer the supernatant to a clean tube spiked with 5 μl of 20% SDS, and immediately shake on a vortex mixer.5. Add 2.5 μl of 20 mg/ml Proteinase K and incubate at 37°C for 15 min.6. Add 400 μl of phenol/chloroform/isoamyl alcohol extraction, centrifuge in a microcentrifuge with oscillation on a vortex mixer, transfer the supernatant to a clean centrifuge tube and repeat the extraction once.
7. Extract with 400 μl of chloroform/isoamyl alcohol and transfer the upper aqueous phase to a clean centrifuge tube.
8. Add 40 μl 3 mol/l sodium acetate buffer (pH 7.5) followed by anhydrous ethanol, mix well and precipitate in dry ice/ethanol for 15-30 min or -20°C overnight.9. Centrifuge at 4°C for 15 min in a microcentrifuge and add 1 ml of 75% ethanol/25% 0.1 mol/l sodium acetate (pH 5.2) to wash the precipitate.
10. After drying, re-dissolve the precipitate with 100 μl of treated water and take 10 μl of RNA diluted in 1 ml of water to measure A280 and A260, which can be stored at -70°C with the RNA.