1. Once the isolation of total RNA is complete, isolation of mRNA can be performed by affinity chromatography on oligo (dT) cellulose, since the vast majority of mRNAs of mammalian cells carry tracts of poly (A) at their 3'-termini. Several companies make kits for preparing poly A+ RNA. We have had success with those by Qiagen (75022 or 70042 [Los Angeles, CA]) and Invitrogen (K1520-02 [San Diego, CA]). We also describe here a reliable protocol that we have been routinely using before changing to the commercially available kit.
2. Equilibrate the oligo (dT) cellulose in sterile loading buffer. Binding affinity is batch-specific. However, a good rule of thumb is that 1 g of resin will bind 2-2.5 mg of poly (A)+ RNA.
3. Pour 1.0 mL packed volume of oligo (dT) cellulose in the Quik-Sep column.
4. Wash the column with, successively, three column volumes of sterile DEPC-treated H2O, 0.1 N NaOH, sterile DEPC-treated H2O. Continue the last wash step until the pH of the column effluent is <8.0.
5. Wash the column with 5 vol of sterile loading buffer.
6. Dissolve the total RNA in sterile water. Heat to 65°C for 5 min. Add an equal amount of 2X loading buffer, and cool the sample to room temperature.
7. Apply the sample to the column, and allow to flow through by gravity. Collect the flow-through.
8. Heat the flow-through to 65°C, cool, and reapply to the column as above.
9. Wash the column with 5-10 column volumes of loading buffer, followed by four column volumes of low-salt loading buffer. The first RNA to elute off the column will be the poly (A)- fraction. The poly (A)+ fraction will elute with the no-salt elution buffer.
10. Elute the poly (A)+ RNA with two to three column volumes of sterile elution buffer. The eluted poly (A)+ RNA can be selected again on oligo (dT)-cellulose by adjusting the NaCl concentration of the eluted RNA to 0.5 M and repeating steps 6-10. We generally elute into Falcon 2059 polypropylene tubes (17 x 100-mm).
11. Add sodium acetate (3 M, pH 5.2) to a final concentration of 0.3 M. Precipitate the RNA with 2.5 vol of 100% ethanol at -20°C overnight.
12. Spin the precipitated RNA at 10,000 rpm (12,000g) for 30 min. Carefully aspirate the supernatant, rinse the pellet with 70% EtOH, and air-dry. Resuspend the RNA pellet in an appropriate volume of DEPC-treated H2O. Determine the RNA concentration spectrophotometrically. Reprecipitate and store as an ethanol precipitate at -20°C. RNA is most stable when stored under ethanol at -70°C.
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