COMPARISON OF 4 METHODS FOR ISOLATING LARGE MESSENGER-RNA - APOLIPOPROTEIN-B MESSENGER-RNA IN BOVINE AND RAT LIVERS

The effectiveness of four methods for isolating large-size mRNA (14 kb ) encoding for hepatic bovine apoprotein B (apo B) was compared. Total RNA of liver (positive controls) and lung (negative controls) samples taken in bovine and rat was extracted using the methods of Chirgwin e f al., Chomczynski and Sacchi, Cathala ct al., and RNAzol, The integri ty of total RNA extracted by the four methods was demonstrated by elec trophoresis on 1% agarose gel and staining with ethidium bromide, Yiel d of extraction was two- to fourfold higher for all samples with the m ethods of Chirgwin ct al. and RNAzol than that with the other methods, By dot blot analysis, apo B mRNA in bovine and rat livers was reveale d preferentially with Chirgwin et al. and RNAzol methods. By Northern blot analysis, a single band corresponding to apo B100 mRNA was shown in bovine and rat livers only with the method of Chirgwin ct al., wher eas a band corresponding to a medium-size mRNA (2.6 kb) encoding for r at phosphoenolpyruvate carboxykinase was revealed in rat liver samples with the four methods. These results showed the favorable role of gua nidium isothiocyanate and guanidium hydrochloride used in the extracti on and the purification of RNA, respectively, especially in the case o f high-molecular-weight and/or low represented RNA such as apo B mRNA in bovine liver. (C) 1996 Academic Press, Inc.