Using Single Molecule RNA FISH to Determine Nuclear Export and Transcription Phenotypes in Drosophila Tissues.
Using Single Molecule RNA FISH to Determine Nuclear Export and Transcription Phenotypes in Drosophila Tissues.
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DOI：
10.1007/978-1-0716-2337-4_8
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发表时间：
2022
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单分子RNA荧光原位杂交（smRNA FISH）是一种广泛应用于检测RNA细胞定位和评估基因表达输出的方法。核孔复合物（NPC）是一种已知的核宏观复合物，既介导核胞质运输，又通过与染色质的相互作用影响转录。因此，NPC蛋白的缺失可能导致mRNA转录或核输出的缺陷。为了区分NPC成分的这两种不同功能，最好同时或在同一细胞中分析转录和mRNA输出。在这里，我们提出了一种smRNA FISH协议，并附带下游自定义MATLAB图像分析，用于果蝇幼虫唾液腺组织。这种方法可以检测核输出和转录表型在同一细胞和作为一个单一的分析，并且可以适应许多其他细胞类型和生物体。
Single molecule RNA fluorescence in situ hybridization (smRNA FISH) is a widely used method for examining cellular localization of RNA and assessing gene expression outputs. The Nuclear Pore Complex (NPC) is a nuclear macro-complex known to both mediate nucleocytoplasmic transport and influence transcription via interactions with chromatin. Consequently, depletion of NPC proteins can result in defects in either transcription or nuclear export of mRNA. To distinguish between these two different functions of NPC components, it is preferable to analyze transcription and mRNA export simultaneously or in the same cell. Here, we present a smRNA FISH protocol with downstream custom MATLAB image analysis for application in Drosophila larval salivary gland tissues. This method can detect both nuclear export and transcriptional phenotypes in the same cell and as a single assay, and can be adapted to many other cell types and organisms.