Qu Yongxia, Boutjdir Mohamed
VA New York Harbor Healthcare System, SUNY Downstate Medical Center, Brooklyn, NY, USA.
Methods Mol Biol. 2007;366:145-58. doi: 10.1007/978-1-59745-030-0_8.
Quantifying the level of mRNA is central to the study of mammalian gene expression. Conventional approaches such as Northern blotting are often prone to low sensitivity and reproducibility. The RNase protection assay (RPA) provides a sensitive alternative for the detection and quantification of mRNA. The RPA is based on the hybridization in solution of a labeled single-stranded antisense RNA probe with a target mRNA. After hybridization, single-strand specific RNases are then used to digest away unhybridized RNA. The hybrid can be resolved by a denaturing gel. Subsequent detection will reveal the appropriate-sized gel band corresponding to the target mRNA. The major advantage of RPA is the high sensitivity and the simultaneous detection and quantification of multiple mRNA targets in a single RNA sample. The primary limitation of RPA is the lack of information on transcript size.
定量mRNA水平是哺乳动物基因表达研究的核心。诸如Northern印迹法等传统方法往往灵敏度低且重复性差。核糖核酸酶保护分析（RPA）为mRNA的检测和定量提供了一种灵敏的替代方法。RPA基于标记的单链反义RNA探针与靶mRNA在溶液中的杂交。杂交后，然后使用单链特异性核糖核酸酶消化未杂交的RNA。杂交体可以通过变性凝胶进行分离。随后的检测将显示与靶mRNA相对应的合适大小的凝胶条带。RPA的主要优点是灵敏度高，并且能够在单个RNA样品中同时检测和定量多个mRNA靶标。RPA的主要局限性在于缺乏转录本大小的信息。
