Gopalakrishna Y, Langley D, Sarkar N
Nucleic Acids Res. 1981 Jul 24;9(14):3545-54. doi: 10.1093/nar/9.14.3545.
A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described. Pulse-labeled RNA isolated by this procedure for Bacillus brevis, Bacillus subtilis, and Escherichia coli B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo(dT)-cellulose. This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA. The presence of polyadenylated RNA in E. coli was confirmed by an independent method which involved hybridization with [3H]polyuridylic acid. Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-treated cells of B. brevis, B. subtilis, and E. coli.
本文描述了一种新的一步法分离细菌RNA的方法，该方法是在十二烷基硫酸钠存在下用蛋白酶K进行裂解。通过该方法分离得到的经脉冲标记的短短芽孢杆菌、枯草芽孢杆菌和大肠杆菌B的RNA，经寡聚（dT）-纤维素吸附测定，发现其中含有相当一部分（15%-40%）的聚腺苷酸化RNA。这与通过苯酚提取法分离的RNA形成对比，苯酚提取法似乎会导致聚腺苷酸化RNA的选择性丢失。通过与[3H]聚尿苷酸杂交的独立方法证实了大肠杆菌中聚腺苷酸化RNA的存在。使用蛋白酶K法分离RNA，可以证明经甲苯处理的短短芽孢杆菌、枯草芽孢杆菌和大肠杆菌细胞能够体外合成聚腺苷酸化RNA。
