Improvements in Immunoprecipitation of Specific Messenger RNA

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Abstract
Procedures for isolation of specific mRNA employing immunoprecipitation of polysomes were previously described. In spite of success with ovalbumin mRNA in the chicken oviduct, there were considerable difficulties in applying these same published techniques to the immunopurification of conalbumin mRNA, despite the fact that the chicken oviduct synthesizes up to 10% of protein as conalbumin. A number of modifications and reinfements are described which proved essential in obtaining intact conalbumin mRNA in high purity and high yields. These reinfements include: improved purification of conalbumin in order to remove contaminating proteins that result in impure antibodies; improved isolation of specific conalbumin antibody in high yields; improved methods for reducing contamination by non-specific polysomes; improved techniques for isolation of RNA from immunoprecipitates resulting in less degradation and higher recovery of conalbumin mRNA; improved techniques for efficient translation of conalbumin mRNA involving treatment of the RNA with methylmercury prior to translation. Problems involved in the immunoprecipitation of different mRNA may differ, and various refinements in techniques may be required for obtaining highly purified preparations of intact mRNA in high yields.