Gruffat D, Piot C, Durand D, Bauchart D
Laboratoire Croissance et Métabolismes des Herbivores, Institut National de la Recherche Agronomique, Centre de Recherches Clermont Ferrand-Theix, Saint Genès Champanelle, France.
Anal Biochem. 1996 Nov 1;242(1):77-83. doi: 10.1006/abio.1996.0431.
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.
比较了四种分离编码牛肝脏载脂蛋白B（apo B）的大尺寸mRNA（14 kb）方法的有效性。采用Chirgwin等人的方法、Chomczynski和Sacchi的方法、Cathala等人的方法以及RNAzol，从牛和大鼠的肝脏（阳性对照）和肺（阴性对照）样本中提取总RNA。通过在1%琼脂糖凝胶上电泳并用溴化乙锭染色，证明了用这四种方法提取的总RNA的完整性。Chirgwin等人的方法和RNAzol对所有样本的提取产量比其他方法高两到四倍。通过斑点印迹分析，Chirgwin等人的方法和RNAzol方法能优先检测到牛和大鼠肝脏中的apo B mRNA。通过Northern印迹分析，仅用Chirgwin等人的方法在牛和大鼠肝脏中显示出一条对应于apo B100 mRNA的条带，而用这四种方法在大鼠肝脏样本中均显示出一条对应于编码大鼠磷酸烯醇丙酮酸羧激酶的中等大小mRNA（2.6 kb）的条带。这些结果表明，异硫氰酸胍和盐酸胍分别在RNA提取和纯化中发挥了有利作用，尤其是在处理如牛肝脏中apo B mRNA这样的高分子量和/或低丰度RNA时。
