Visualisation of ribosomal RNA (rRNA) synthesis in eukaryotic cells in culture
Visualisation of ribosomal RNA (rRNA) synthesis in eukaryotic cells in culture
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DOI：
10.1023/a:1009752804126
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发表时间：
1998-01-01
期刊：
影响因子：
--
通讯作者：
Kalland, Karl-Henning
中科院分区：
文献类型：
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作者：
Haukenes, Gunnar;Kalland, Karl-Henning
当引入培养的真核细胞中时，核糖核苷酸类似物5-溴尿苷5 '-三磷酸（BrUTP）取代UTP掺入RNA中。用多聚甲醛固定后，可以通过免疫学方法检测到新生溴标记的RNA聚合酶II转录物（前mRNA），而没有看到核仁RNA聚合酶I转录物（rRNA）的标记。我们描述了一个快速，方便的程序，可视化BrUTP取代rRNA在培养细胞。BrUTP的摄取是通过在暴露于盖玻片上的细胞之前将其与转染试剂FuGENETM 6络合来介导的，从而消除了对细胞的显微注射或透化的需要。通过使用变性固定剂如甲醇、乙醇或丙酮代替甲醛，核仁rRNA的溴标记变得容易检测。
When introduced into eukaryotic cells in culture, the ribonucleotide analogue 5-bromouridine 5'-triphosphate (BrUTP) becomes incorporated into RNA instead of UTP. After fixation with paraformaldehyde nascent bromine labelled RNA polymerase II transcripts (pre-mRNA) can be detected by immunological methods, while no labelling of nucleolar RNA polymerase I transcripts (rRNA) is seen. We describe a rapid and convenient procedure for visualisation of BrUTP-substituted rRNA in cells in culture. Uptake of BrUTP is mediated by complexing it with the transfection reagent FuGENETM 6 before exposure to cells on coverslips thus eliminating the need for microinjection or permeabilisation of the cells. By using denaturing fixatives such as methanol, ethanol or acetone instead of formaldehyde bromine labelling of nucleolar rRNA becomes readily detectable.