Venkatesh Lakshminarayan K, Fasina Olufemi, Pintel David J
Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri-Columbia, Columbia, MO, USA.
Methods Mol Biol. 2012;883:121-9. doi: 10.1007/978-1-61779-839-9_9.
The ribonuclease protection assay (RPA) has emerged as an important methodology for the detection, mapping, and quantification of RNAs. In this assay, total or cytoplasmic RNAs are hybridized to a high-specific activity antisense radioactive RNA probe synthesized by in vitro transcription from the SP6 or T7 promoter of an appropriate linearized plasmid template by the bacteriophage SP6 or T7 polymerase, respectively. The RNA hybrids are subjected to RNAse digestion and the protected products are resolved by denaturing polyacrylamide gel electrophoresis to allow detection of specific RNA fragments by subsequent autoradiography. RPAs are highly sensitive, the probes can be specifically targeted, and, when performed in probe excess, are quantitative, making them the method of choice for many analyses of RNA processing events.
核糖核酸酶保护分析（RPA）已成为检测、定位和定量RNA的重要方法。在该分析中，总RNA或细胞质RNA与通过噬菌体SP6或T7聚合酶分别从合适的线性化质粒模板的SP6或T7启动子进行体外转录合成的高特异性活性反义放射性RNA探针杂交。RNA杂交体经过RNA酶消化，受保护的产物通过变性聚丙烯酰胺凝胶电泳进行分离，以便通过随后的放射自显影检测特定的RNA片段。RPA高度灵敏，探针可特异性靶向，并且在探针过量的情况下进行时具有定量性，使其成为许多RNA加工事件分析的首选方法。
