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RESEARCH DIVISION Laboratory Manual

 


 

RNAse Protection Assay

  1. Cut 10ug of plasmid DNA where you want the transcript to end.
  2. Add an equal volume of 2X PK buffer:
    200mM TrisHCl pH 7.5
    25mM EDTA
    300mM NaCl
    2% SDS
    200ug/ml proteinase K
  3. 37C for 30min and then extract with phenol/CHCl3 and ethanol precipitate. Resuspend at 1mg/ml in DDW.
  4. Labelling reaction:
    2ul 5X SP6 buffer: 200mM TrisHCl, 30mM MgCl2, 10mM spermadine
    0.5ul RNAsin
    0.5ul 10mM rATP
    0.5ul 10mM rCTP
    0.5ul 10mM rGTP
    5ul a-P32-UTP (400Ci/mMol)
    0.5ul template
    0.5ul 200mM DTT
    0.5ul appropriate RNA polymerase
    Incubate for 90-120min at 41C.
  5. Add 0.5ul RNAsin and 0.5ul RNAse free DNAse (5mg/ml). Incubate for 15min at 37C.
  6. Add 10ug tRNA and 100ul DDW. Phenol/CHCl3 extract and precipitate with 50ul 5M NH4 acetate, 400ul ethanol.
  7. Resuspend in 100ul 1X hybridization buffer:
    0.1 volume 10X hyb. (400mM pipes, pH 6.4, 4M NaCl, 10mM EDTA)
    0.1 volume DDW
    0.8 volume deionized formamide.
    1ul should be 1000-2000 cps on the mini monitor.
  8. Spin down RNA as an ethanol ppte and resuspend in 24ul 1X hyb. buffer and add 0.5-1.0ul of probe (from step 7). Include a control RNA eg. tRNA or negative tissue.
  9. Incubate overnight at 37-50C.
  10. Add 350ul RNAse buffer to hybridization and vortex immediately.
    1X RNAse buffer:
    10mM Tris pH 7.5
    5mM EDTA
    300mM NaCl
    40ug/ml RNAse A (Sigma)
    2ug/ml RNAse T1 (Sigma)
  11. Incubate at ___ C for ____min.

Optimal times and temperatures vary for different probes. Overdigestion gives lots of small bands and underdigestion gives large artifactual bands. 37C for 10-15min works well in many cases.

  1. To stop the reaction add 20ul 10% SDS and 5ul 20mg/ml proteinase K. Incubate for 15min 37C.
  2. Add 2-10ug tRNA carrier. Phenol chloroform extract and ethanol precipitate without additional salt. Air dry pellet and resuspend in 3-4ul sequencing loading buffer and run on a sequencing gel after boiling.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998