Femino A M, Fay F S, Fogarty K, Singer R H
Department of Anatomy and Structural Biology and Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Science. 1998 Apr 24;280(5363):585-90. doi: 10.1126/science.280.5363.585.
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.
荧光原位杂交（FISH）和数字成像显微镜技术经过改进，以实现对单个RNA分子的检测。合成了每个分子带有五种荧光染料的寡脱氧核苷酸探针，并对单个探针发出的光进行了校准。杂交细胞的彻底解卷积图像中的光点给出了与单个信使RNA分子一致的荧光强度和探针之间的距离。血清诱导后β-肌动蛋白转录位点的分析揭示了单个基因的同步和周期性转录。转录起始和终止的速率以及信使RNA加工过程可以通过沿着转录单元定位探针来确定。这种方法扩展了FISH的能力，能够在单个细胞上产生定量的分子信息。
