Isolation of cytoplasmic RNA from mammalian cells
Isolation of cytoplasmic RNA from mammalian cells
The following method is a simple and effective method for purifying cytoplasmic RNA from tissue culture cells, which appeared at an earlier stage of RNA isolation, with centrifugation to remove the nucleus as the main step.
Operation method
Isolation of cytoplasmic RNA from mammalian cells
Materials and Instruments
Ice-cold phosphate buffer NaCl KCl Na2HP04-7H20 KH2PO4 Ice-cold lysis buffer: LTris-HCl NaCl MgCL2 NkmidetP-40 Placenta RNase inhibitor SDS Protease K Phenol Chloroform Isoamyl alcohol Sodium ethanolate (DEPC-treated) Anhydrous ethanol Ethanol Sodium acetate DEPC-treated water Move I. Materials and equipment Caveat 1 ) This method can process 2 X10 at a time.7cells per 107This method can treat 2 X107 cells at a time, obtaining 30~100ug RNA per 10 7 cells. The A260/080should be between 1.7 and 2.0. 0.2 ) Be careful when resuspending the cells in the second step to avoid foaming, and wait until the suspension clears quickly to show that cell lysis is complete.3 ) If the collected cells have been transfected with DNA, the contaminating DNA can be removed by digestion with RNase-free DNase I, and the RNA can be recovered by extraction with phenol:chloroform:isoamyl alcohol and chloroform:isoamyl alcohol as described above. For more product details, please visit Aladdin Scientific website.
Beckman JS 4. 2 Rotor or other similar equipment
1) Ice-cold phosphate buffer solution (PBS):137 mmol/L NaCl, 2.7 mmol/l KCl,4.3 mmol/l Na2 HP04-7 H20,1.4 mmoI/L KH2PO4pH7.3
2) Ice-cold lysis buffer: 50 mmol/L Tris-HCl,pH 8.0 l00 mmoL/L NaCl,5 mmoL/L MgCl2 ), 0.5% (V/V) NkmidetP-40 1000 U/ml placenta, RNase inhibitor (e.g. RNasin) and 1 mmol/LDTT, prepared in DEPC-treated water. water, filtered for sterilization
3) 20% SDS
4) 20 mg/ml Proteinase K
5) Phenol: chloroform: isoamyl alcohol (25:24:1)
6) 3mol/L sodium acetate (DEPC treated) pH 5.2
7) Anhydrous ethanol
8) 75% ethanol/25% 0.lmoL/L sodium acetate, pH 5.2
9) DEPC treated water
II Methods of Operation
1) Collect cells: ①For walled cells, wash every 10 cm dish with Iml ice-cold PBS three times, then scrape off the cells with a small volume of PBS and transfer to centrifuge tube (put on ice) 300 g centrifugation for 5 min to remove the upper part of the wall.
②For suspension cells, centrifuge the cells at 300 g for 5 min and remove the supernatant. Resuspend the cells with half volume of ice-cold PBS, centrifuge as before and remove the supernatant.
2) Resuspend cells with 375ul of ice-cold lysis buffer and incubate on ice for 5 min. centrifuge for 2 min at _4°C in a microcentrifuge. Transfer the supernatant to a tube containing 4ul of 20% SDS and mix immediately with shaking.
(3) Add 2.5ul of 20 mg/ml proteinase K and incubate at 37℃ for 5 min.
4) Add 400ul of phenol: chloroform: isoamyl alcohol, shake Imin. centrifuge, transfer the upper aqueous phase to a clean tube, repeat the extraction once, and then 400ul of chloroform: isoamyl alcohol extraction once. The upper aqueous phase was transferred to a clean tube.
5) Add 40ul3mol/l sodium acetate (pH5.2) and lml anhydrous ethanol, mix, and precipitate on ice; precipitate for 15~30 min or at 20℃ overnight.
6) Centrifuge at 1200 g for 15 min at 4°C. Wash the precipitate with 1 ml of 75% ethanol/25½0.lmol/1 sodium acetate (pH5.2). After drying, dissolve in 100ul of DEPC water. A small portion was taken out to determine its concentration and purity, and the rest was stored at minus 70℃.