Comparison of four methods for isolating large mRNA: apolipoprotein B mRNA 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 et al., Chomczynski and Sacchi, Cathala et al., and RNAzol. The integrity of total RNA extracted by the four methods was demonstrated by electrophoresis on 1% agarose gel and staining with ethidium bromide. Yield of extraction was two- to four-fold higher for all samples with the methods of Chirgwin et al. and RNAzol than that with the other methods. By dot blot analysis, apo B mRNA in bovine and rat livers was revealed 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 et al., whereas a band corresponding to a medium-size mRNA (2.6 kb) encoding for rat phosphoenolpyruvate carboxykinase was revealed in rat liver samples with the four methods. These results showed the favorable role of guanidium isothiocyanate and guanidium hydrochloride used in the extraction and the purification of RNA, respectively, especially in the case of high-molecular-weight and/or low-represented RNA such as apo B mRNA in bovine liver.
Gruffat
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D
Laboratoire Croissance et Métabolismes des Herbivores, Institut National de la Recherche Agronomique, Centre de Recherches Clermont Ferrand-Theix, Saint Genès Champanelle, France.
Piot
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C
Durand
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D
Bauchart
D
D
D000818:Animals; D001055:Apolipoproteins B / Q000032:analysis* / Q000235:genetics*; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D019791:Guanidine; D006146:Guanidines; D008099:Liver / Q000737:chemistry*; D008168:Lung / Q000737:chemistry; D008970:Molecular Weight; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats
D001055:Apolipoproteins B; D006146:Guanidines; D012333:RNA, Messenger; D019791:Guanidine