Demonstration of tissue-specific gene expression by in situ hybridization.
Demonstration of tissue-specific gene expression by in situ hybridization.
复制标题
通过原位杂交展示组织特异性基因表达。
DOI：
10.1016/0076-6879(87)52071-7
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发表时间：
1987
影响因子：
--
通讯作者：
Angerer,RC
中科院分区：
文献类型：
--
作者：
Angerer,LM;Cox,KH;Angerer,RC
原位杂交已成为鉴定表达特定基因的单个细胞的一种有价值的工具。最近，方法已经变得足够灵敏，可以检测每个细胞中仅存在几个分子水平的mrna。当mrna仅在混合群体中的一小部分细胞中表达时，原位杂交可能是最敏感的核酸杂交技术。作者的实验室已经证明，反义RNA探针提供了一种独特的优势组合，用于原位杂交检测单个mrna。最重要的是，反义探针提供了一个很大的灵敏度增加，由于缺乏竞争探针的自我重新关联。RNA-RNA双链的高稳定性允许使用更高的杂交后洗涤温度来实现给定的碱基配对保真度（严格性），这也减少了背景。RNA转录本可以从截断的模板合成，这样它们基本上没有载体序列。这种序列纯度最大化的信噪比，因为较低的探针浓度需要饱和的目标rna。
In situ hybridization has emerged as a valuable tool for the identification of individual cells expressing specific genes. Recently, methods have become sufficiently sensitive to detect mRNAs present at the level of only a few molecules per cell. When mRNAs are expressed in only a small fraction of the cells in a mixed population, in situ hybridization may be the most sensitive nucleic acid hybridization technique available. The authors' laboratory has shown that antisense RNA probes offer a unique combination of advantages for detection of individual mRNAs by in situ hybridization. Most importantly, antisense probes provide a large increase in sensitivity due to the absence of competing probe self-reassociation. The high stability of RNA-RNA duplexes allows use of higher post hybridization wash temperatures to achieve a given fidelity of base pairing (stringency), which also reduced backgrounds. RNA transcripts can be synthesized from truncated templates such that they are essentially devoid of vector sequences. This sequence purity maximizes the signal to noise ratio since lower probe concentrations are required to saturate target RNAs.