Adjuvants to immunological methods for mRNA purification. Application to the isolation of mRNA for carbonic anhydrase I from rabbit reticulocytes.
In order to generate the molecular probes needed to investigate the seemingly coordinate expression of carbonic anhydrase (CA I) and beta-globin within erythrocytes during human development, CA I-containing polyribosomes have been isolated from rabbit reticulocytes by reaction with purified antibodies to CA I followed by immunoadsorption of immune complexes with formalin-fixed protein A-bearing bacteria. In the course of such isolation, a series of maneuvers were seen to have a markedly favorable influence on the level of purity attained. These maneuvers include the use of 5 mg/ml of heparin concentrations to attenuate nonspecific binding and entrapment of unwanted polyribosomes, the addition of 200 units/ml of placental ribonuclease inhibitor to augment recovery in reactions which by test already appeared RNase-free, and the preadsorption of polyribosomes with formalin-fixed bacteria prior to immunological reaction so as to remove a subset of polyribosomes seemingly predisposed to nonspecific binding. In the absence of all of the maneuvers, attained purity was no greater than a few per cent. When all were employed, CA I-mRNA derived from immunopurified polyribosomes was recovered with more than 80% purity and 20% yield, as evident from both immunoassays and electrophoresis of its cell-free products.
D000818:Animals; D002256:Carbonic Anhydrases / Q000235:genetics*; D006493:Heparin / Q000494:pharmacology; D007158:Immunologic Techniques; D008722:Methods; D011132:Polyribosomes / Q000187:drug effects / Q000201:enzymology; D012333:RNA, Messenger / Q000302:isolation & purification*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*
D012333:RNA, Messenger; D006493:Heparin; D002256:Carbonic Anhydrases