Oligo(dT)纤维素层析分离Poly(A)+RNA

Selection of Poly(A)⁺ RNA by Oligo(dT)-Cellulose Chromatography

• Ethanol

• Ethanol (70%)

• NaCl (5 M)

• NaOH (10 N)

• Elution buffer

• 10 mM Tris-Cl (pH 7.6)

• 1 mM EDTA(pH 8.0)

• 0.05% SDS
   
  The stock solutions of Tris-Cl and EDTAused to make elution buffer should be freshly autoclaved (15 minutes at 15 psi [1.05 kg/cm²] on liquid cycle) and then diluted with the appropriate amount of sterile DEPC-treated H²O. Then add the SDSfrom a concentrated stock solution (10% or 20%) made in sterile DEPC-treated H²O. IMPORTANT:Do not attempt to sterilize elution buffer by autoclaving because it froths excessively.

• 2x Oligo(dT)-cellulose loading buffer

• 40 mM Tris-Cl (pH 7.6)

• 1 M NaCl

• 2 mM EDTA (pH 8.0)

• 0.2% (w/v) sodium lauryl sarcosinate

• Prepare as described below. 
   
  Make up Tris-Cl (pH 7.6) from a fresh bottle in autoclaved, DEPC-treated H₂O. Prepare NaCl and EDTA in 0.1% DEPC in H₂O. Store for at least 12 hours at 37°C and autoclave the mixture for 15 minutes at 15 psi (1.05 kg/cm²) on liquid cycle. To prepare sterile loading buffer, mix appropriate amounts of RNase-free stock solutions of Tris-Cl (pH 7.6), NaCl, and EDTA (pH 8.0) in an RNase-free container. Allow the solution to cool to approximately 65°C and then add sodium lauryl sarcosinate from a 10% stock solution that has been heated to 65°C for 30 minutes. 
   
  Alternatively, substitute 0.05 M sodium citrate for Tris-Cl and treat the sodium citrate/NaCl/ EDTA mixture and sodium lauryl sarcosinate with DEPC(please see Protocol 7.1). Store loading buffer at room temperature.

• Sodium acetate (3 M, pH 5.2)

• Sterile, DEPC-treated H₂O (see MC3, p. 7.84)

• Total cellular RNA

• Sorvall SS-34 rotor (4°C)

• Cuvettes, for measuring absorbance at 260 nm

• Dispo column or pasteur pipette plugged with sterile glass wool
   
  Before use, treat Dispo column with DEPC. Sterilize the plugged pasteur pipette by baking for 4 hours at 300°C.

• Oligo(dT)-cellulose, usually purchased commercially as a dried powder
   
  Columns of oligo(dT)-cellulose can be stored at 4°C and reused many times. Between uses, the column should be regenerated by sequential washing with NaOH, H₂O, and loading buffer as described in Steps 1-3 of this protocol. Spun columns of oligo(dT)-cellulose are available from several manufacturers.

• pH paper (pH test strips)

• Water bath (65°C)

1. Suspend 0.5-1.0 g of oligo(dT)-cellulose in 0.1 N NaOH
    
   

2. 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.
    
   

3. 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.
    
   

4. 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. 
    
   

5. 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.
    
   

6. 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.
    
   

7. 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).
    
   

8. 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.
    
   

9. Precipitate the fractions containing a majority of the OD₂₆₀ material by the addition of 2.5 volumes of ethanol at -20°C.
    
   

10. 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.
     
    

11. 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.
     
    

12. 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. 
     
    

13. 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.
     
    

14. 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.
     
    

15. 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.
     
    

16. Store the preparation of poly(A)⁺ RNA.