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Oligo(dT)-cellulose is the method of choice for the isolation of poly(A)-containing RNA from polysomal or total RNA preparations. The matrix is stable, relatively resistant to nuclease activity, and does not shed polynucleotides that might inhibit cDNA synthesis. In addition, the column is run at room temperature, which allows easy monitoring by flow specrophotometry without the need of a cooling jacket. It should be noted, however, that cellulose can non-specifically bind DNA. Therefore, the samples applied to the column should contain little or no DNA, which might be released with the poly(A)-contaIning RNA.
Use autoclaved or DEPC treated glassware, virgin plasticware, and RNase-free solutions. Since the column is run at room temperature, strict RNase-free technique must be used if intact poly(A)-containing RNA is to be obtained.


  1. Materials

    1. Oligo(dT)-cellulose, type II (Collaborative Research, Inc.)

    2. 0.1N NaOH
      4.00 grams per liter

    3. CRAB (per 500ml)
      final conc.
      14.61 grams NaCl 0.5M
      0.6057 grams Tris-OH 10mM
      2.50 grams SDS 0.5%
      0.1905 grams EDTA 1mM
      500ml pH 7.5

    4. CREB (per 500ml)
      final conc.
      0.6057 grams Tris-OH 10mM
      0.2500 grams SDS 0.05%
      0.1905 grams EDTA 1mM
      500ml pH 7.5

  2. Methods

    1. Sample preparation:

      1. EtOH precipitate about 100 A260 units (3-4 mg) of the RNA preparation by adding 1/10 volume of 2.5M NaOAc and 2.5 volumes of cold EtOH. Store overnight at -20C.
      2. Centrifuge at 14KRPM for 30 min at 4C in a Sorval SS-34 rotor. Discard the supernatant and dry the pellet under vacuum.
      3. Dissolve the pellet in 10ml CRAB. Store frozen (-20C).

    2. Column preparation: Perform all steps at room temperature.

      1. Suspend 1.0 gram of oligo(dT)-cellulose (use a flamed spatula to weigh it out) in 10ml CREB.
      2. Pour into an autoclaved 10ml syringe column. The column outflow tube should be connected to a flow-cell. Rinse any remaining cellulose into the column with CREB.
      3. Pass 25ml of CREB through the column by gravity (the flow rate should be about 1/2 drop/sec.), then equilibrate the column with 25ml CRAB.
      4. Warm up the UV spectrophotometer, and place the flow cuvette in the holder. Be sure the cuvette is in the proper position so that light beam passes through the flow chamber being used.
      5. Be sure the machine is on 'narrow aperture', the wavelength is set at 260nm, and set the recorder to a calabrated absorbance of 3.0, a chart speed of 10 cm/hr, and zero both the spectrophotometer and the chart.

    3. Chromatography: Perform all steps at room temperature.

      1. Lower the buffer (CRAB) level to just above the bed surface, and apply the sample to the column.
      2. Allow the sample to pass through the column at about 15 drops/min. Collect the outflow in an Oak Ridge tube.
      3. When the entire sample has run through the column and the fluid level reaches the surface of the bed, rinse the sides of the column with 1ml of CRAB, and allow that to run into the column so that the buffer level is again lowered to the surface of the bed.
      4. Gently fill the column with CRAB, so that the bed is not disturbed. As this wash runs into the column, keep adding more CRAB until the column has been washed with 20ml of CRAB. After the absorbance decreases to background, the flow rate can be increased to about 1/2 drop/sec.
      5. Lower the buffer level to the bed surface. Add 1ml of CREB directly to the bed, being careful not to disturb the bed. Allow this to run into the column at a flow rate of about 15 sec/drop. When the CREB level reaches the bed surface, add another 1ml to the bed, and allow this to pass into the column.
      6. When the buffer reaches the bed surface, gently fill the syringe with CREB and allow this to run through the column. When the A260 begins to increase, start collecting the effluent, and continue to collect until the A260 approaches baseline.
      7. Wash the column with an additional 10ml of CREB at about 1/2 drop/sec., then clamp off the outlet tube.
      8. Add 1/10 volume of 2.5M Na acetate and 2.5 volumes of cold EtOH to the collected A260 absorbing elution peak, and store overnight at -20C. Pellet the poly(A)-containing RNA at 16KRPM for 1 hr at 4C in an SW27 or SW27.1 rotor.
      9. Discard the supernatant, and invert the tube over a Kim-wipe for about 15 min. to allow the tube to drain-dry. Dry the pellet under vacuum and dissolve in ddH2O to a concentration of 1ug/ul. (the yeild of poly(A)-containing RNA can be calculated from the known collected volume and the area under the elution A260 peak, assuming that an A260 of 1 = 38.5 ug RNA/ml)

    4. CLEANING THE COLUMN

      1. After each use, pass 10ml 0.1N NaOH through the column, then wash the column with ddH20 until the effluent has a neutral pH.

    5. STORAGE

      1. Short-term storage:

        If the column is to be used the next day, then re-equilibrate with 20ml CREB, and store at room temperature. Begin chromatography at step 3 of COLUMN PREPARATION, where the column is washed with CREB. The same procedures are used for storage of up to a week, except that the column is stored at 4C.

      2. Long-term storage:

        If the column is not needed for a week or more, the matrix should be stored as an EtOH powder. After cleaning the column with NaOH and rinsing with ddH2O, wash the column with 25ml EtOH. Using a small weighing spatula, transfer the matrix to a plastic scintillation vial (rinse out the column with a small amount of EtOH to recover it all) and lyophilize overnight. Store at -70C.

Aviv and Leder 1977 PNAS 69(6):1408-1412
Collaborative Research Data Sheet: Oligo(dT)-cellulose, type II
Maniatis,T., Fritsch,E,F., and Sambrook,J. 1982 'Molecular Cloning' CSH pages 197-198

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