Characterization of messenger RNA by direct translation from agarose gels.
A method for characterizing nanogram quantities of poly(A)-containing messenger RNAs that have been fractionated according to size by electrophoresis through agarose gels has been developed. The mRNAs from Friend leukemia cells were identified by the protein products they encode, as determined by slicing the agarose gel and directly translating the enclosed mRNA with an extract from rabbit reticulocytes that had been treated with micrococcal nuclease. A number of parameters which affect the efficiency of translation in this system have been examined. These include the sensitivity of the in vitro translational system to RNA, the agarose concentration, the incubation temperature, and the addition of either exogeneous tRNA or RNasin. The procedure is rapid, simple, reproducible, and applicable for the fractionation and characterization of mRNAs from any source.
D000818:Animals; D002474:Cell-Free System; D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008836:Micrococcal Nuclease; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification; D012333:RNA, Messenger / Q000302:isolation & purification*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D011506:Proteins; D012333:RNA, Messenger; D008836:Micrococcal Nuclease