Isolation of undegraded free and membrane-bound polysomal mRNA from rat brain.
A new procedure is described for the isolation of undegraded free and membrane-bound polysomal mRNA from rat brain in which polysomes are simultaneously separated from soluble components of the cell and slowly sedimenting ribosome species and concentrated by rate-zonal centrifugation on short linear sucrose gradients. This avoids the problem encountered in conventional procedures of pelleting polysomes along with membranous materials that are not solubilized by detergents and that appear to contain traces of nucleases. It also permits a more thorough analysis of the mRNA populations actively engaged in protein synthesis, since both polyadenylated and nonpolyadenylated mRNAs are isolated together. Moreover, the likelihood of sedimenting nonpolysomal mRNP particles along with polysomes is reduced by using rate-zonal rather than pelleting centrifugation. The translational activity in vitro of free and membrane-bound polysomal RNA prepared in this way is high and is about 1.5 times that of RNA prepared by a conventional pelleting technique. The difference is attributable to better preservation of large mRNAs, as inferred from two-dimensional gel electrophoretic analysis of translation product abundance. The recovery of both classes of polysomal RNA is about 90%. The method is simple, efficient, and adapted for isolation of small amounts of polysomal RNA.
D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D002501:Centrifugation, Zonal; D005470:Fluorometry; D011132:Polyribosomes / Q000032:analysis*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D012333:RNA, Messenger