Cytoplasmic RNA extraction experimental procedure

Because of the many sources of RNA, the extraction methods are different. Generally, there are phenol method, detergent method and guanidine hydrochloride method, among which phenol method is the most commonly used. The tissue was homogenized and centrifuged with phenol. The RNA was dissolved in the upper phenol-saturated aqueous phase. After adding cold ethanol to the aqueous phase, the RNA was precipitated as a white flocculent. This method preferably removes DNA and protein and obtains biologically active RNA.

experimental method
  • Basic plan
Experimental Materials
RNA
Reagents, kits
PBS cell lysate SDS proteinase K phenol chloroform isoamyl alcohol absolute ethanol
Instruments, consumables
Centrifuge incubator
Experimental procedure
1. For single-layer cultured cells

(1) Cells cultured per 10 cm culture dish were washed 3 times with ice-cold PBS.

(2) 1 ml each time and 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 it.

(2) The cells were resuspended in 1/2 original volume of ice, and then centrifuged and discarded.
3. Resuspend the cells in 375 μl of ice-cold cell lysis buffer and place in an ice bath for 5 min.

4. Centrifuge at 4 °C for 2 min in a microfuge and transfer the supernatant to a clean tube with 5 μl of 20% SDS and immediately shake on the vortex mixer.
5. Add 2.5 μl of 20 mg/ml proteinase K and incubate for 15 min at 37 °C.
6. Add 400 μl of phenol/chloroform/isoamyl alcohol extraction, shake on a vortex mixer, centrifuge with a microcentrifuge, transfer the supernatant to a clean centrifuge tube, and repeat extraction for 1 time.

7. Draw in 400 μl chloroform/isoamyl alcohol and transfer the upper aqueous phase to a clean centrifuge tube.

8. Add 40 μl of 3 mol/l sodium acetate buffer (pH 7.5), add absolute ethanol, mix 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 precipitate.

10. After drying, reconstitute the pellet with 100 μl of treated water, dilute 10 μl of RNA in 1 ml of water, measure A 280 and A 260 , and store the RNA at -70 °C.

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