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RNA molecules can be 3' end-labeled using [5'-32P]pCp (cytidine-3',5'-bis-phosphate) and RNA ligase. The RNA to be labeled must have a 3' hydroxyl, so most degradation products will not label with pCp (except the original 3'-end, of course). pCp end-labeling is the method of choice for end labeling stable RNAs because, in most cases, the 5' ends of such molecules are hybridized the the 3' ends of the molecules & are recessed, so that they label poorly with 32P-ATP & polynucleotide kinase. This is particularly true of tRNA molecules.
This procedure includes two methods (take your choice)for the synthesis of 32P-pCp, because of the enormous cost of the commercial product (about $1 per uCi). The compound is easy to synthesis, & is fresher than the commercial product.
Remember to use RNase-free technique whenever handling RNAs! In addition, the amounts of radioisotope used in the synthesis of pCp are large - be particularly careful, & check afterwards to be sure you've cleaned-up all your hot-spots.

  1. Materials

    1. 5X pCp synthesis buffer
      125mM potassium CHES, pH9.5
      25mM MgCl2
      0.25% NP40

    2. 2X pCp buffer
      100mM HEPES, pH7.5
      30mM MgCl2
      20% DMSO

    3. TE-SDS
      10mM Tris, pH8
      1mM EDTA
      0.1% SDS

    4. Column dye
      0.25% bromophenol blue 50 mg
      50% glycerol 10ml
      1/2 X TE-SDS 10ml 1X
      20ml

  2. Methods

    1. Synthesis of [5'-32P]pCp (method #1)

      1. Working on ice, mix:
        25ul 100mM Tris/20mM MgCl2, pH8
        5ul 50mM DTT
        5ul 15mM spermine-HCl
        10ul 750uM cytidine-3'-phosphate
        5ul ATP (gamma-32P) - about 1mCi
        2ul polynucleotide kinase (about 20u)

      2. Incubate 37C overnight.
      3. Heat for 3min. at 90C, then centrifuge 5min. Recover the supernatant & store at -20C.

    2. Synthesis of [5'-32P]pCp (method #2)

      1. Working on ice, mix:
        2ul 5X pCp synthesis buffer
        3ul ddH2O
        4ul 10mM 3'-CMP
        1ul (10-30 units) T4 polynucleotide kinase

      2. With a pipette gun set to 1ul, draw 1ul of the mixture into a pipette tip, then expel the liquid back into the tube. Wash the "empty" pipette tip in 20ul ddH2O by drawing & expelling the liquid back & forth in the pipette tip. Save this (on ice) as a "before" sample of the reaction.
      3. Incubate 3hr 37C.
      4. Repeat step 2 for an "after" sample of the reaction.
      5. Add 90ul ddH2O to the reaction mixture, then heat at 90C for 5 min. Store at -20C.
      6. Spot the "before" and "after" samples about 1cm from the bottom of a 3 x 10cm PEI plate, & develop in 550mM (NH4)2SO4 (it'll take about 2hr). Autoradiograph for 15min. without screens. The predominant spot in the "before" sample (ATP) should all or mostly disappear in the "after" sample, being replaced by a spot that migrates nearly with the solvent front (pCp).

    3. 3' end-labeling RNA

      1. Dry down the RNA to be labeled in an eppendorf tube. 5ug is good.
      2. Working on ice, mix the following in the tube containing dried RNA:
        25ul 2X pCp buffer
        1ul 1mM ATP
        1ul 1mg/ml BSA (nuclease-free)
        4ul 100mM DTT
        10ul [5'-32P]pCp
        8ul DEPC-treated ddH2O
        2.5ul RNA ligase

      3. Incubate at 4C overnight.
      4. The labeled RNA can then be gel purifed (if you want to separate RNAs from the labeled mix) or G50 column chromatography (if you want all of the labeled RNAs).

    4. Column chromatography

      1. Add 50ul TE-SDS and 10ul column dye to the sample.
      2. Apply the sample onto a 0.7 X 20 cm G50 sephadex column in TE-SDS.
      3. Run the column with TE-SDS, collecting the first 20 fractions, 12 drops each. Wash the column in TE-SDS until the blue dye is all washed out of the column.
      4. Spot 2ul of each fraction onto 4-5mm squares of 3MM paper. Count each square in 4ml scintillation fluid.
      5. Pool the G50 excluded peak (i.e. the first peak), which usually is at fractions 7-10. Store at -20C until use.

Elizabeth S. Haas, personal communication
Charles J. Daniels, personal communication

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