In situ reverse transcription for detection of hybridization between oligonucleotides and their intracellular targets.
In situ reverse transcription for detection of hybridization between oligonucleotides and their intracellular targets.
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原位逆转录用于检测寡核苷酸与其细胞内靶标之间的杂交。
DOI：
10.1006/meth.1999.0785
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Singer,RH
中科院分区：
文献类型：
--
作者：
Politz,JC;Singer,RH
重要的是要知道，反义治疗引起的表型变化是因为反义分子与其细胞内靶点特异性杂交，而不是通过某种非特异性的间接途径。我们在这里描述了一种方法，可以用来检测反义寡核苷酸与其细胞内靶RNA的杂交，并进一步识别杂交物位于原位的位置。寡核苷酸首先被活细胞摄取，然后细胞被固定并进行原位逆转录反应。逆转录实验利用了这样一个事实，即只有与RNA杂交的寡核苷酸才能作为逆转录酶的引物，并允许将标记的核苷酸掺入cdna；未杂交的寡核苷酸不会启动逆转录。我们通过比较在含有和不含有阳离子脂类的情况下摄取寡聚(DT)的细胞中与Poly(A)RNA杂交的寡聚(DT)的水平来说明这种方法。
It is often important to know that a phenotypic change caused by antisense treatment has occurred because the antisense molecule has specifically hybridized to its intracellular target, rather than by some nonspecific, indirect route. We describe here a method that can be used to detect hybridization of an antisense oligodeoxynucleotide to its intracellular target RNA and, furthermore, to identify the sites at which hybrids are located in situ. Oligodeoxynucleotides are first taken up by the live cell and then cells are fixed and subjected to an in situ reverse transcription reaction. The reverse transcription assay exploits the fact that only oligonucleotides that are hybridized to RNA will act as primers for reverse transcriptase and allow incorporation of labeled nucleotide into cDNA; unhybridized oligonucleotides will not prime reverse transcription. We illustrate this approach by comparing the levels of oligo(dT) hybridized to poly(A) RNA in cells that have taken up the oligo(dT) with and without cationic lipid in the medium.
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影响因子：
64.5
作者：
LENK, R;RANSOM, L;PENMAN, S
通讯作者：
PENMAN, S
mRNA的形成和功能
DOI：
--
发表时间：
1997
期刊：
影响因子：
--
作者：
J. Richter
通讯作者：
J. Richter
影响因子：
14.9
作者：
AGRAWAL, S;CHRISTODOULOU, C;GAIT, MJ
通讯作者：
GAIT, MJ
DOI：
10.1073/pnas.95.11.6043
发表时间：
1998-05-26
影响因子：
11.1
作者：
Politz, JC;Browne, ES;Pederson, T
通讯作者：
Pederson, T
5-(N-氨基己基)氨基甲酰基-2-脱氧尿苷对核酸内切酶稳定性和寡脱氧核苷酸激活 RNase H 能力的影响。
影响因子：
14.9
作者：
Y. Ueno;I. Kumagai;N. Haginoya;A. Matsuda
通讯作者：
A. Matsuda