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@Oligo(dT) cellulose chromatography can be performed either by the column or centrifuge method. For the preparative isolation of unlabeled eukaryotic poly(A)-containing RNA, the column method is preferred because of its ease and because it is easy to scale up. For the quantitative analysis of radiolabeled poly(A)-containing RNAs (or the isolation of labeled poly(A)-containing RNA from non-eukaryotes) the centrifuge method is often better, because 1) it can be performed at 4C rather than 25C, increasing the ability to retain RNAs with short polyadenylation, 2) because it is easier to run on a micro scale, 3) because fewer fractions are collected, reducing handling, 4) because a finer matrix can be used (i.e. type III) for quantitative binding, and 5) because the method is faster.
@Remember, use sterile and RNase-free technique at all times to prevent sample degradation. Autoclave all solutions, and weight the oligo(dT) cellulose with a flamed and cooled spatula.

  1. Reagents

    1. A & W buffer (per liter)
      @ final conc.
      29.1 grams NaCl 0.5M
      1.21 grams Tris, pH7.5 10mM

    2. Elution buffer (per liter)
      @ final conc.
      1.21 grams Tris, pH7.5 10mM

    3. 5M NaCl
      @58.44 grams per 200ml

  2. Methods

    1. In a sterile 1.5ml eppendorf microfuge tube, mix the RNA sample (from 1-5 ml culture) and 0.11 volumes 5M NaCl. Adjust the volume to 1ml with A&W buffer.
    2. Add 17mg oligo(dT) cellulose per ml culture extracted from, and incubate with vigorous enough shaking to keep the oligo(dT) cellulose suspended at 4C for 1 hr.
    3. Working on ice, pellet the matrix in a clinical centrifuge at 1000Xg for 1 min., collect the supernatant, and wash the pellet with 1ml aliquots of ice cold A&W buffer. The oligo(dT) cellulose should be washed 8 - 10X.
    4. After the final A&W wash, resuspend the pellet in 1ml EB at 45C, and incubate for 15min. at 45C with frequent shaking. Pellet as before and repeat the 45C elution.
    5. Wash the oligo(dT) cellulose twice more with 45C EB, without the 45C incubation.
    6. Resuspend the final pellet in 1ml EB.
    7. Count each supernatant fraction, as well as the oligo(dT) cellulose suspension, in 9ml Aquasol 2.

      NOTE:
      @If this, or the column method, is to be used analytically on radiolabeled RNA that has not been EtOH precipitated (or any other method that removes unincorporated material), such as an SDS-protease K lysate, the sample should be pre-assayed for percent TCA precipitatablity, as follows:

      1. Spot 1 - 5ul RNA sample onto a glassfiber filter, and allow this to air-dry. This is the total CPM test.
      2. Mix the same volume of RNA sample with 10ul 2m/ml sonicated salmon sperm DNA (in TE) and 500ul ice cold 5% TCA. Incubate 5-15 min. on ice.
      3. Gently filter through a glassfiber filter. Wash the filter by filtering 5ml ice cold 5% TCA.
      4. Air-dry the filter (use a infrared lamp, if you're in a hurry), and count both the total and TCA pptable CPM filters in 5ml scintillation fluid.
      5. TCA pptable CPM/total CPM X 100 = % TCA insoluable
      6. When calculating % poly(A)-containing RNA from a run, remember to multiply the % retained by % TCA insoluable to get the % TCA insoluable retained.


Gopalakrishna, et al 1981 NAR \9(14)\:3545
J W Brown (TCA \ppt\ion modification)

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