Cytoplasmic DNA can be detected by RNA fluorescence in situ hybridization
Cytoplasmic DNA can be detected by RNA fluorescence in situ hybridization
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DOI：
10.1093/nar/gkz645
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发表时间：
2019-10-10
影响因子：
14.9
通讯作者：
Shav-Tal, Yaron
中科院分区：
文献类型：
--
作者：
Greenberg, Eliraz;Hochberg-Laufer, Hodaya;Shav-Tal, Yaron
荧光原位杂交（FISH）可用于DNA或RNA分子的细胞内检测。通过DNA FISH检测DNA序列需要DNA双螺旋的变性以允许荧光探针与单链形式的DNA杂交。这些杂交条件需要高温和低pH，这可能会破坏RNA，因此RNA通常不能通过DNA FISH检测到。相反，RNA FISH不需要变性步骤，因为RNA是单链的，因此DNA分子不能通过RNA FISH检测。因此，DNA FISH和RNA FISH是相互排斥的。在这项研究中，我们表明，瞬时转染到细胞中的质粒DNA是很容易检测到的细胞质中的RNA FISH，而不需要变性，转染后不久，几个小时。然而，质粒通常不能在细胞核中检测到，除非质粒被有效地导入细胞核，这可能意味着转染后DNA的包装状态更开放。在细胞质中质粒DNA的检测对RNA FISH实验具有影响，并打开了一个窗口，以研究DNA存在于细胞质中的条件。
Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.