核酸酶保护分析
核酸酶保护分析（英語：Nuclease protection assay，亦称为核糖核酸酶保护测定）是一项用于生物化学与遗传学的实验室技术，可用以从细胞中提取出来混杂的RNA样本中鉴定出单独的RNA。此技术可以在即使总浓度很低的情况下鉴定一种或多种已知序列的RNA分子。被提取出来的RNA首先与反义RNA或DNA探针相混合，探针是与被研究的序列相互补的，接下来互补链相互杂交以形成双股RNA（或DNA-RNA杂交分子）。此混合物接下来接触核糖核酸酶，这种酶特异性地切割仅为单股的RNA，但对双股RNA来说没有作用。当反应进行完全时，易受影响的RNA片段被降解为非常短的低聚物或单个核苷酸；存留下来的RNA片段是与之前所加进的反义链相互补的RNA，因此包含目的序列。
探针
[编辑]The probes are prepared by cloning part of the gene of interest in a vector under the control of any of the following promoters, SP6, T7 or T3. These promoters are recognized by DNA dependent RNA polymerases originally characterized from bacteriophages. The probes produced are radioactive as they are prepared by in vitro transcription using radioactive UTPs. Uncomplemented DNA or RNA is cleaved off by nucleases. When the probe is a DNA molecule, S1 nuclease is used; when the probe is RNA, any single-strand-specific ribonuclease can be used. Thus the surviving probe-mRNA complement is simply detected by autoradiography.
用途
[编辑]Nuclease protection assays are used to map 内含子s and 5' and 3' ends of transcribed 基因 regions. Quantitative results can be obtained regarding the amount of the target RNA present in the original cellular extract - if the target is a 信使RNA, this can indicate the level of 转录 of the gene in the cell.
They are also used to detect the presence of double stranded RNA, presence of which could mean RNA interference.
RNA印迹 is a laboratory technique that produces similar information. It is slower and less quantitative, but also produces accurate information about the size of the target RNA. Nuclease protection assay products are limited to the size of the initial probes due to the destruction of the non-hybridized RNA during the nuclease digestion step.