Visualization of single RNA transcripts in situ
Visualization of single RNA transcripts in situ
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DOI：
10.1126/science.280.5363.585
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发表时间：
1998-04-24
期刊：
影响因子：
56.9
通讯作者：
Singer, RH
中科院分区：
文献类型：
--
作者：
Femino, A;Fay, FS;Singer, RH
修改荧光原位杂交（FISH）和数字成像显微镜，以检测单个RNA分子。每个分子与五个荧光染料合成寡脱氧核苷酸探针，并校准单个探针发出的光。杂交细胞的详尽反卷曲图像中的光点产生了荧光强度和与单信使RNA分子一致的探针之间的距离。血清诱导后β-肌动蛋白转录位点的分析表明，单基因的同步和周期性转录。转录启动和终止和使者RNA处理的速率可以通过沿着转录单元的定位探针来确定。这种方法扩展了鱼类在单个细胞上产生定量分子信息的力量。
Fluorescence in situ hybridization (FISH) and digital imaging microscopy were modified to allow detection of single RNA molecules. Oligodeoxynucleotide probes were synthesized with five fluorochromes per molecule, and the light emitted by a single probe was calibrated. Points of light in exhaustively deconvolved images of hybridized cells gave fluorescent intensities and distances between probes consistent with single messenger RNA molecules. Analysis of beta-actin transcription sites after serum induction revealed synchronous and cyclical transcription from single genes. The rates of transcription initiation and termination and messenger RNA processing could be determined by positioning probes along the transcription unit. This approach extends the power of FISH to yield quantitative molecular information on a single cell.