Suspend 0.5-1.0 g of oligo(dT)-cellulose in 0.1 N NaOH
Pour a column of oligo(dT)-cellulose (0.5-1.0-ml packed volume) in a DEPC-treated Dispo column (or a pasteur pipette, plugged with sterile glass wool and sterilized by baking for 4 hours at 300°C). Wash the column with 3 column volumes of sterile DEPC-treated H2O.
Wash the column with sterile 1x oligo(dT)-cellulose-loading buffer (dilute from 2x stock using sterile DEPC-treated H2O) until the pH of the effluent is <8.0. Use pH paper for this measurement.
Dissolve the RNA in sterile H2O and heat the solution to 65°C for 5 minutes. Cool the solution to room temperature quickly and add 1 volume of 2x oligo(dT)-cellulose-loading buffer.
Apply the solution of RNA to the column and, in a sterile tube, immediately begin to collect the material flowing through the column. When all of the RNA solution has entered the column, wash the column with 1 column volume of 1x oligo(dT)-cellulose-loading buffer while continuing to collect the flow-through.
When all of the liquid has emerged from the column, heat the collected flow-through to 65°C for 5 minutes and reapply it to the top of the column. Again collect the material flowing through the column.
Wash the column with 5-10 column volumes of 1x oligo(dT)-cellulose-loading buffer, collecting 1-ml fractions into sterile plastic tubes (e.g., microfuge tubes).
Use quartz or disposable methacrylate cuvettes to measure the absorbance at 260 nm of a 1:20 dilution of each fraction collected from the column using 1x oligo(dT)-cellulose loading buffer as a blank.
Precipitate the fractions containing a majority of the OD260 material by the addition of 2.5 volumes of ethanol at -20°C.
Elute the poly(A)+ RNA from the oligo(dT)-cellulose with 2-3 column volumes of sterile, RNase-free elution buffer. Collect fractions equivalent in size to one third of the column volume.
Use quartz or disposable methacrylate cuvettes to measure the absorbance at 260 nm of each fraction collected from the column. Pool the fractions containing the eluted RNA.
To purify poly(A)+ RNA further, heat the preparation of RNA to 65°C for 3 minutes and then cool it quickly to room temperature. Adjust the concentration of NaCl in the eluted RNA to 0.5 M using 5 M NaCl and carry out a second round of chromatography on the same column of oligo(dT)-cellulose (i.e., repeat Steps 3 and 5-11).
The material obtained after a single round of chromatography on oligo(dT)-cellulose usually contains approximately equal amounts of polyadenylated and nonpolyadenylated species of RNA. Polyadenylated RNA may be further purified as described by a second round of chromatography on oligo(dT)-cellulose.
To the poly(A)+ RNA eluted from the second round of oligo(dT)-cellulose chromatography, add 3 M sodium acetate (pH 5.2) to a final concentration of 0.3 M. Mix well. Add 2.5 volumes of ice-cold ethanol, mix, and store the solution for at least 30 minutes on ice.
Recover the poly(A)+ RNA by centrifugation at 10,000g (9000 rpm in a Sorvall SS-34 rotor) for 15 minutes at 4°C. Carefully discard the supernatant, and wash the pellet (which is often invisible) with 70% ethanol. Recentrifuge briefly, remove the supernatant by aspiration, and store the open tube in an inverted position for a few minutes to allow most of the residual ethanol to evaporate. Do not allow the pellet to dry.
Redissolve the damp pellet of RNA in a small volume of sterile, DEPC-treated H2O. Use quartz or disposable methacrylate cuvettes to measure the absorbance at 260 nm of each fraction collected from the column. Pool the fractions that contain RNA.
Store the preparation of poly(A)+ RNA.