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œœœœœ@Primer extension analysis of RNA 5' ends@œœœœœ



The sites of transcription initiation or RNA processing are generally determined by S1 protection or primer extension (PE) analysis. Although S1 protection must be used for 3'-end analysis, 5'-end analysis is usually more easily & precisely done by primer extension analysis. In this method, a short antisense 5' end-labeled DNA primer (usually a synthetic oligonucleotide, but sometimes a small restriction fragment) is hybridized to RNA, usually total cellular RNA, then DNA is synthesized from this primer using reverse transcriptase. RTase will copy the RNA from the site of primer annealing to the 5'-end of the RNA molecule. The reactions are then analyzed by electrophoresis in sequencing gels in lanes adjacent to Sanger sequencing reactions of DNA containing the gene of question, using the same primer as that used in the PE analysis. The transcription initiation site (usually) can then easily be identified as the band in the sequencing reaction directly parallel to the run-off reverse transcript. Multiple transcription initiation sites will appear as multiple bands in the PE lane. RNA processing sites (i.e. in the case of stable RNAs) will also appear in PE analyses, and must be distinguished from transcription initiation sites by other means (usually by examination of the DNA sequence for promoter elements).
Before you begin, of course, you must have the sequence of the region in question and a DNA primer in a region that must be in the transcript, i.e. the upstream part of the ORF or structural RNA. Usually, the primers are 18-25 base synthetic oligonucleotides. The method described is unsuitable for eukaryotic RNAs, unless it is known that no introns (which would give misleading results) are present between the 5' end of the RNA & the primer. You must also pre-make sequencing ladders, for standards, of the region being analyzed. These are easily prepared from cloned DNA using the same primer used in the PE analysis and either sequenase or klenow chain termination sequencing procedures. Make enough for several gels, or , if you prefer, prpare the sequencing ladders fresh for each PE experiment (this is probably best, if you know the sequencing works well each time). Lastly, you will need RNA. Total cellular RNA is OK - it should be undegraded and purifed free of DNA, for example by the CsCl/EtBr method. You'll need alot of RNA - 125ug per lane on the gel, or 250ug per experiment. The RNA is probably the most important ingredient of the experiment - good RNA, good results. Remember, use RNase-free technique whenever handling RNA!

  1. Materials

    1. 10X Kinase buffer
      0.5M Tris,pH9.5
      0.1M MgCl2
      50mM DTT
      50% glycerol

    2. Hybridization buffer
      0.4M NaCl
      400mM PIPES, pH6.4
      1mM EDTA
      80% (v/v) formamide

    3. Phenol:chloroform
      48% chloroform
      50% TE sat'd phenol
      2% isoamyl alcohol

    4. PIPES/NaCl
      10mM PIPES, pH6.4
      400mM NaCl

    5. S1 buffer
      50mM Tris, pH 8.2
      10mM DTT
      6mM MgCl2
      25ug/ml actinomycin D
      500uM dATP
      500uM dGTP
      500uM dTTP
      500uM dCTP

    6. Sequencing sample buffer
      80% formamide
      10mM NaOH
      1mM EDTA
      0.1% xylene cyanol FF
      0.1% bromophenol blue

  2. Methods

    1. 5' end-labeling of the oligonucleotide primer

      (If you're using a DNA restriction fragmnt as a primer, 5' end-label this DNA at the downstream end as usual for Maxam & Gilbert sequencing.)

      1. Working on ice, mix:
        10-15 picomoles oligonucleotide primer
        (~5ul of a 1:10 dil'n of primer if prepared as suggested)
        5ul 10X kinase buffer
        100uCi gamma-32P-ATP
        to 49u ddH2O
        1ul (10-20u) polynucleotide kinase

      2. Incubate for 1hr at 37C.
      3. Add 10ul column dye (or agarose gel loading dye) to the sample.
      4. Apply the sample onto a 0.7 X 20 cm G50 sephadex column in ddH2O & run the column with ddH2O, collecting the first 20 fractions, 12 drops each. Wash the column in ddH2O until the blue dye is all washed out of the column.
      5. Spot 2ul of each fraction onto 4-5mm squares of 3MM paper. Count each square in 4ml scintillation fluid.
      6. Pool the G50 excluded peak (i.e. the first peak), which usually is at fractions 7-10. Store at -20C until use.

    2. Primer extension analysis

      1. Prepare 3 eppendorf tubes with the following ingredients:
        tube #1 tube #2 tube#3
        125ug RNA + + -
        100KCPM primer + + +

      2. Add 1/4th volume of 2M ammonium acetate and 3 volumes of EtOH and incubate 10 min. in a dry ice:EtOH bath, then centrifuge 10 min. Drain the pellets completely (draw out away droplets of liquid with a drawn-out grass capillary pipette) and dry in a rotovac.
      3. Dissolve the pellet in 30ul hybridization buffer. This is a critical step, and difficult. Pipette the liquid up & down, batter the pellet with the pipette tip, etc, for 10-20 min. The RNA MUST be in solution! If the pellet doesn't seem to want to dissolve, heat at 80C for 5 min., then pipette up & down, etc, to dissolve.
      4. Incubate for 10 min. at 80C in a water bath or heating block, then turn the bath/block to the hybridization temperature. It should take 30 min. to 2 hr for then temperture change to take place. Incubate ON at the hybridization temperature. The hybridization temperature is determined by the following formula:

        Hybridization T (in C) = 29.3 + 0.41(%G+C of the primer)

      5. Add:
        45ul 2M ammonium aceate
        145ul ddH2O
        800ul EtOH
        then incubate 10 min. in a dry ice:EtOH bath, centrifuge 10 min., discard the liquid and add 1ml ice cold 70% EtOH. Chill and centrifuge as before, drain the pellets and dry in a rotovap.

      6. Dissolve the pellets in 10ul PIPES/NaCl, again being careful to thoroughly dissolve the RNA by rapid pipetting, vortexing & physically smashing the pellets. (Don't try to dissolve the pellets by heating, of course!)
      7. Add 80ul S1 buffer & mix well by pipetting. Add 5 units of AMV reverse transcriptase and incubate at 42C for 1hr.
      8. Add 9ul 3M Na acetate and 100ul phenol:chloroform and vortex. Centrifuge 2min. and collect the upper, aqueous, phase & add 250ul EtOH. Chill 10min. in dry ice:EtOH & centrifuge 10min. Discard the liquid and dry the pellet in a rotovac. Dissolve the pellet (each one) in 10ul sequencing sample dye.
      9. Before electrophoresis, heat the samples at 90C for 5min. & quick-chill on ice. Electrophoresis should be in a 6% sequencing gel agjacent to sequencing reactions of cloned DNA using the same oliogonucleotide primer & chain termination sequencing reactions.
      10. After electrophoresis, dry the gel & autoradiograph as usual.


Transcription & Translation (Hames & Higgins, eds) IRL Press
JWBrown, unpublished data
GSBeckler, PhD thesis

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