核酸酶保護分析
核酸酶保護分析（英語：Nuclease protection assay，亦稱為核糖核酸酶保護測定）是一項用於生物化學與遺傳學的實驗室技術，可用以從細胞中提取出來混雜的RNA樣本中鑑定出單獨的RNA。此技術可以在即使總濃度很低的情況下鑑定一種或多種已知序列的RNA分子。被提取出來的RNA首先與反義RNA或DNA探針相混合，探針是與被研究的序列相互補的，接下來互補鏈相互雜交以形成雙股RNA（或DNA-RNA雜交分子）。此混合物接下來接觸核糖核酸酶，這種酶特異性地切割僅為單股的RNA，但對雙股RNA來說沒有作用。當反應進行完全時，易受影響的RNA片段被降解為非常短的低聚物或單個核苷酸；存留下來的RNA片段是與之前所加進的反義鏈相互補的RNA，因此包含目的序列。
探針
[編輯]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
[編輯]Nuclease protection assays are used to map 內含子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 轉錄 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印跡 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.