Humphreys-Beher M G, King F K, Bunnel B, Brody B
Biotechnol Appl Biochem. 1986 Oct;8(5):392-403.
Biologically active RNA has been isolated from several tissues implicated in the pathogenesis of the genetic disease cystic fibrosis. To ensure that mRNA from stomach and pancreas has not been degraded, it must be isolated within 2 h postmortem, while intact RNA can be isolated from lung material up to 20 h postmortem. It is imperative that tissue removed during autopsy be dispersed in a strong denaturant buffer (guanidine isothiocyanate) to inactivate nucleases. While stomach and pancreas yields of RNA are low, relative to the amount of gland material, yields of RNA from lung samples are sufficient for the further isolation of mRNA by oligo(dT)-cellulose chromatography. The biological integrity of subsequently isolated mRNA has been assessed by AMV-reverse transcriptase cDNA synthesis as well as by in vitro translation. Protein products generated in this manner show distinct differences from mRNA translation patterns of age- and sex-matched controls when compared with mRNA from CF samples. R0t analysis of cDNA libraries thus generated indicates that the complexity of such libraries is representative of the starting RNA species.
已从与遗传性疾病囊性纤维化发病机制相关的多个组织中分离出具有生物活性的RNA。为确保胃和胰腺的mRNA未被降解，必须在死后2小时内进行分离，而完整的RNA可从死后长达20小时的肺组织材料中分离出来。尸检期间取出的组织必须分散在强变性缓冲液（异硫氰酸胍）中以灭活核酸酶，这一点至关重要。相对于腺体材料的量，胃和胰腺的RNA产量较低，而肺样本的RNA产量足以通过寡聚（dT）-纤维素层析进一步分离mRNA。随后分离出的mRNA的生物学完整性已通过禽成髓细胞瘤病毒逆转录酶cDNA合成以及体外翻译进行了评估。与来自囊性纤维化（CF）样本的mRNA相比，以这种方式产生的蛋白质产物与年龄和性别匹配的对照的mRNA翻译模式显示出明显差异。由此产生的cDNA文库的R0t分析表明，此类文库的复杂性代表了起始RNA种类。
