Kornblum J S, Projan S J, Moghazeh S L, Novick R P
Department of Plasmid Biology, Public Health Research Institute, New York, NY 10016.
Gene. 1988;63(1):75-85. doi: 10.1016/0378-1119(88)90547-1.
We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.
我们开发了一种快速定量特定细菌RNA种类的方法。该方法可测量RNA的稳态水平，在超过16倍的RNA浓度范围内产生线性响应，并且可用于金黄色葡萄球菌、大肠杆菌和枯草芽孢杆菌。在此方法中，将约7×10⁸个生物体的剪切全细胞裂解物（制备方法同质粒筛选）在琼脂糖上分离，转移至硝酸纤维素滤膜上，与放射性标记的DNA探针杂交，然后进行放射自显影。通过对滤膜上的放射性条带计数来定量RNA种类。我们已将该方法应用于测量编码β-内酰胺酶、ermC rRNA甲基化酶和β-半乳糖苷酶α-互补片段的基因的mRNA诱导情况。诱导后，观察到每个基因的mRNA增加了10倍。β-内酰胺酶的mRNA峰值水平在30分钟后出现，β-半乳糖苷酶在20分钟后出现，ermC rRNA甲基化酶在5分钟后出现。
