Most mammalian messenger RNAs (mRNAs) carry a poly(A)+ tail, and Poly(A)+-carrying RNAs can be isolated from total RNA using this protocol.
Operation method
Oligodeoxythymidine cellulose purification band poly( A)+RNA
Materials and Instruments
NaOH. oligodeoxythymidine (oligo(dT) ] cellulose DEPC-treated water 1X spiking buffer Elution buffer NaAc (pH 5.2) Ethanol TE buffer. Move I MATERIALS AND EQUIPMENT Caveat 1 ) The maximum sample volume of 1 ml of oligo(dT)-cellulose is 10 mg of total RNA, if the amount of total RNA is less, the amount of oligo((dT)-cellulose should be reduced . To avoid loss of poly( A)ˉ RNA during column passage and subsequent steps. It also avoids waste.2 ) Heating the RNA can destroy the secondary structure of possible poly( A)ˉ tails .3 ) About 1%~2% of total RNA can be recovered from the spiked sample.¾ of the total RNA can be recovered from the total RNA spiked.puly(A)ˉ RNA from 10 to 7% of the total RNA spiked.71~5ug of Poly( A)ˉ RNA can be obtained from 10 7 mammalian cells.+RNA.RNA from 10 7 mammalian cells.4) Electrophoretic detection of poly( A)+ RNA should be below 20k h and diffuse.RNA should be below 20k h and diffuse, and most of them are concentrated in the range of 5~10kb.5) Sodium dodecyl sarcosinate is insoluble, if the room temperature is lower than 18℃, it may hinder the flow of liquid in the column, lithium chloride can be used instead of sodium chloride in the spiking buffer to solve this problem. For more product details, please visit Aladdin Scientific website.
1 ) 5 mol/L NaOH
2 ) Oligodeoxythymidine (oligo(dT) ] cellulose
3) DEPC-treated water
4) 1X spiking buffer: 20 mmol/L Tris-CL PH (6.7). 0.5 mol/L NaCl, l mmol/L EDTA ( pH 8. 0 ). 0.1% sodium dodecyl sarcosinate
5 ) Elution buffer: 10 mmol/L Tris-Cl (PH7.6), 1 mmol/L EDTA (PH 8.0), 0.05% SDS
6) 3 mol/ L NaAc (pH5.2)
7 ) Ethanol
8) TE buffer
II Methods of operation
1) Firstly, wash the silica chromatography columns with 10 ml of 5 mol/L NaOH and then rinse with water. The column was first washed with 10 ml of 5mol/L NaOH and then rinsed with water. Take 0.5 g of oligo(df) cellulose powder and add it to 1m]0.1mol/LNaOH, pour it into the column, and rinse it with about 10 ml of water. (The column can be made of siliconized Pasteur pipette plugged with siliconized glass wool or a siliconized disposable column with a capacity of 2 ml.
2) Equilibrate the column with 10-20 ℃ spiking buffer until the pH of the effluent solution is about 7.5.
3) Heat an aqueous solution containing 2 mg of total RNA at 70 ℃ for 10 min, cool it quickly to room temperature, add an equal volume of 2X spiking buffer, top up the sample and collect the effluent solution with a sterile cuvette immediately. Collect the effluent immediately with a sterile test tube. Wash with double the column volume of spiking buffer, incubate the effluent at 70℃ for 5 min, and reapply to the column twice.
4) Wash the column with 5~10 times the column volume of spiking buffer, collect the effluent step by step, 1 ml each, and measure the OD260 of each collection tube. Elute until the OD260 is very small or zero.
5) Elute Poly(A)ˉ RNA with 2~3 times the column volume of elution buffer, and then elute with 1/3~1/2 of the column volume of elution buffer, then elute with 1/3 to 1/3 of the column volume of elution buffer. Collect the eluate in steps of 1/3 to 1/2 column volume.
6) Adjust the concentration of sodium acetate in the collected KNA solution to 0.3mol/L, add 2.5 times the volume of ethanol, and leave at 20°C overnight or in dry ice/ethanol for 30 min.
7) Centrifuge the precipitate of RNA at 10,000 g at 4°C for 15mim. Discard the ethanol, and then wash the precipitate with 70% ethanol. Discard the 70% ethanol, dry the precipitate, and redissolve it in a small amount of RNAase-free TE buffer. After heating at 70°C for 5 min, the RNA quality was checked by 1% agarose gel electrophoresis.
S)poly(A )+1RNA solution can be stored directly at 70°C or at -70°C with 3 times the volume of ethanol.