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œœœœœ@End-labeling RNA@œœœœœ



@RNA samples can be 5' or 3' end-labeled using commercially available materials. 3' end-labeling is carried out by RNA ligase, which 'ligates' a single labeled nucleotide to the 3'-OH end of the RNA. The labeled substrate in a 3',5'-bis-phosphate (usually cytidine 5',3'-bis-phosphate, 32P-pCp), so that the resulting 3' end is a phosphate, and no longer a substrate for the enzyme. 5' end-labeling is carried out exactly as for DNA (see that procedure in M&M as well), unless the 5' ends are capped; in that case, the RNA must first be de-capped with tobacco acid pyrophosphatase (TAP). Naturally, RNase-free technique MUST be used at all times, including wearing gloves at all times, using sterile autoclaved or DEPC treated materials and high-purity chemicals.

  1. MATERIALS FOR 3' END LABELING

    1. 10mM Tris-HCl, pH7.4

    2. 5X RNA ligase buffer
      250mM HEPES, pH8.3
      50mM MgCl2
      16.5mM DTT
      0.5mM ATP

    3. 10X GE tracking dye (as for glyoxal gels)

    4. 10mM Tris-HCl, pH7.6

  2. Materials of 5' end-labeling

    1. 10X TAP buffer
      0.5M Na acetate, pH6
      0.1M 2-mercaptoethanol

    2. 0.5M Tris-HCl, pH8.3 at 37C

    3. 250mM KH2PO4, pH9.5 (with KOH)

    4. 250mM MgCl2

    5. 50mM dithiothreitol

    6. 10mM Tris-HCl, pH7.6

  3. PROCEDURE FOR 3' END-LABELING

    1. Working on ice, mix the following:
      6ul 5X RNA ligase buffer
      3ul DMSO
      3.5ul glycerol
      5ul (50uCi)32P-pCp
      1ul (0.5-1.0ug) RNA
      9ul ddH2O
      2.5ul (10 units) RNA ligase
      30ul @

    2. Incubate for 24hr at 4C, then stop the reaction at 65C for 5 min.
    3. Add 60ul 10mM Tris and 10ul 10X GE tracking dye. Apply the sample to a 6x150mm G-50 sephadex column, in 10mM Tris. Collect 35 12drop fractions.
    4. Spot 5ul from each fraction to a filter paper square, and count in 5ml Aquasol.
    5. Pool the void volume peak CPM fractions, and store frozen.

  4. PROCEDURE FOR 5' END-LABELING CAPPED RNAs

    1. Mix the following ingredients, on ice:
      1ul 10X TAP buffer
      Xul RNA (up to 1ug)
      Yul ddH2O
      1ul TAP (2-5 units)
      10ul final volume

    2. Incubate 37C for 30 min, then add:
      2ul 0.5M Tris-HCl, pH8.3
      7ul ddH2O
      1ul CIAP (or BAP)(4 units)

    3. Incubate at 56C (or 37C for BAP) for 30 min, then add 1ul K-PO4, pH9.5 & mix.
    4. Transfer the mixture to a fresh tube containing 20-50uCi gamma32P-ATP dried down in the tube. Add 1ul 250mM MgCl2, 2ul 50mM DTT and 1ul (4-10 units) polynucleotide kinase.
    5. Incubate 37C for 30 min. Add 5ul 10X GE tracking dye and 20ul 10mM Tris-HCl, pH7.6. Apply the sample to a 6x150mm G-50 column (in 10mM Tris, pH7.6), and collect 35 12drop fractions. Spot 2ul samples of each fraction onto filter paper squares and count them in 5ml Aquasol.
    6. Pool the void volume peak CPM fractions and store frozen.

      NOTE: For use with non-capped RNAs, mix the RNA with 2ul 0.5M Tris-HCl, pH7.6 + 0.1M 2-ME, 1ul CIAP and ddH2O to 20ul, then start at step 3.


England 1978 Nature 275:560
Brown 1985 J.Bacteriol. in press
Efstratiadis 1977 NAR 4:4165

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