FISHing on a Budget.
FISHing on a Budget.
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按预算钓鱼。
DOI：
10.1007/978-1-0716-1585-0_5
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Kim,HaroldD
中科院分区：
文献类型：
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作者：
Wadsworth,GableM;Kim,HaroldD
通过荧光原位杂交(FISH)对RNA转录本进行灵敏的定量是理解单细胞中定量基因表达的一个普遍存在的部分。有许多技术可以在细胞内识别和定位转录本，但它们往往成本高昂，劳动密集型。在这里，我们提出了一种使用单标记短DNA寡核苷酸探针在酵母细胞中进行FISH的方法。这种方法对于目标长度有限(200个核苷酸)的高度受限的鱼类应用是有效的。这种方法可以定量不同的RNA异构体或使用荧光共振能量转移(FRET)来检测相邻序列块的共转录。由于这种方法依赖于单探针，因此它也比多探针标记策略更具成本效益。
Sensitive quantification of RNA transcripts via fluorescence in situ hybridization (FISH) is a ubiquitous part of understanding quantitative gene expression in single cells. Many techniques exist to identify and localize transcripts inside the cell, but often they are costly and labor intensive. Here we present a method to use a singly labeled short DNA oligo probe to perform FISH in yeast cells. This method is effective for highly constrained FISH applications where the target length is limited (<200 nucleotides). This method can quantify different RNA isoforms or enable the use of fluorescence resonance energy transfer (FRET) to detect co-transcription of neighboring sequence blocks. Since this method relies on a single probe, it is also more cost-effective than a multiple probe labeling strategy.