Improved method for the purification of urokinase m-RNA

Abstract

A major approach to the investigation of differential gene expression and regulation of protein synthesis in animal cells is through the study of messenger RNA. Recently, intense efforts have centered on the isolation, characterization, and quantitation of the m-RNAs coding for specific proteins. Therefore, the purification method of m-RNA must be improved. Human Embryonic Kidney(HEK) cells were cultured in MEM media. Total RNA was extracted from HEK cells by CsCl centrifugation method or selective method, then passed through oligo-d(T) cellulose column to obtain total m-RNA. Messenger activity of the preparation was checked in vitro using the rabbit reticulocyte lyste (RRL) system and the peptides synthesized in vitro were separated on SDS-polyacrylamide gel electrophoresis(PAGE). Identification of urokinase(UK)-like material was proved by immunoprecipitation with antibody raised against highly purified UK. Purity of total RNA prepared by CsCl centrifugation method was 1.6 and maximum absorbance peak appered 260nm. But purity of total RNA prepared by selective method was 1.4 and maximum absorbance peak appeared 270nm. Purity of m-RNA produced by CsCl centrifugation method was 2.0 and maximum absorbance peak appeared 260nm. But purity of m-RNA produced by selective method was 1.69 and maximum absorbance peak appeared 260nm. Quantitation of m-RNA produced by CsCl centrifugation method was 243ug. And quantitation of m-RNA produced by selective method was 177ug. In conclusion, it was found that CsCl centrifugation method appeared to be superior to the selective method interms of simplicity, processing time, yield, intactness, and biological activity of resulting RNA. And autoradiography showed that m-RNA coded for urokinase-like material.