Turbo FISH: a method for rapid single molecule RNA FISH.
Turbo FISH: a method for rapid single molecule RNA FISH.
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DOI：
10.1371/journal.pone.0075120
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Raj A
中科院分区：
文献类型：
--
作者：
Shaffer SM;Wu MT;Levesque MJ;Raj A
RNA荧光原位杂交技术（RNA FISH）的进步使从业者能够通过荧光显微镜检测单个细胞中的单个RNA分子，从而实现高度准确和敏感的基因表达定量。然而，目前的方法通常使用杂交时间在2-16小时左右，限制了其在快速诊断等应用中的潜力。我们在这里提出了一套RNA FISH（称为Turbo RNA FISH）的条件，使我们能够在不超过5分钟的杂交时间和3分钟的洗涤时间内进行准确的测量，并表明杂交时间可以低至30秒，同时仍然产生可量化的图像。我们进一步表明，快速杂交与我们最近开发的RNA FISH的iceFISH和SNP FISH变体兼容，它们分别能够进行染色体和单碱基区分。我们的方法简单且成本有效，并且有可能大大提高RNA FISH的吞吐量和适用范围。
Advances in RNA fluorescence in situ hybridization (RNA FISH) have allowed practitioners to detect individual RNA molecules in single cells via fluorescence microscopy, enabling highly accurate and sensitive quantification of gene expression. However, current methods typically employ hybridization times on the order of 2–16 hours, limiting its potential in applications like rapid diagnostics. We present here a set of conditions for RNA FISH (dubbed Turbo RNA FISH) that allow us to make accurate measurements with no more than 5 minutes of hybridization time and 3 minutes of washing, and show that hybridization times can go as low as 30 seconds while still producing quantifiable images. We further show that rapid hybridization is compatible with our recently developed iceFISH and SNP FISH variants of RNA FISH that enable chromosome and single base discrimination, respectively. Our method is simple and cost effective, and has the potential to dramatically increase the throughput and realm of applicability of RNA FISH.
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作者：
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DOI：
10.1073/pnas.1206069109
发表时间：
2012-06-05
影响因子：
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作者：
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通讯作者：
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