RNA. 2014 Feb;20(2):260-6. doi: 10.1261/rna.041905.113. Epub 2013 Dec 17.
The ability to detect RNA molecules in situ has long had important applications for molecular biological studies. Enzyme or dye-labeled antisense in vitro runoff transcripts and synthetic oligodeoxynucleotides (ODN) both have a proven track record of success, but each of these also has scientific and practical drawbacks and limitations to its use. We devised a means to use commercially synthesized oligonucleotides as RNA-FISH probes without further modification and show that such probes work well for detection of RNA in cultured cells. This approach can bind a high concentration of fluorescent ODN to a short stretch of an RNA using commercial DNA synthesis outlets available to any laboratory. We call this approach for creating in situ hybridization probes Fluorescence In Situ Hybridization with Sequential Tethered and Intertwined ODN Complexes (FISH-STICs). We demonstrate that one FISH-STIC probe can detect mRNA molecules in culture, and that probe detection can be improved by the addition of multiple probes that can be easily adapted for robust mRNA quantification. Using FISH-STICs, we demonstrate a nonoverlapping distribution for β-actin and γ-actin mRNA in cultured fibroblasts, and the detection of neuron-specific transcripts within cultured primary hippocampal neurons.
长期以来，检测 RNA 分子的原位能力在分子生物学研究中具有重要的应用。酶或染料标记的体外转录物的反义链和合成的寡脱氧核苷酸（ODN）都具有成功的记录，但每种方法都有科学和实际的缺点，并且在使用上存在限制。我们设计了一种方法，无需进一步修饰即可将商业合成的寡核苷酸用作 RNA-FISH 探针，并证明这些探针在检测培养细胞中的 RNA 时效果良好。这种方法可以使用任何实验室都可以使用的商业 DNA 合成器，将高浓度的荧光 ODN 结合到 RNA 的短链上。我们将这种创建原位杂交探针的方法称为使用顺序连接和交织的寡核苷酸复合物的荧光原位杂交（FISH-STIC）。我们证明，一个 FISH-STIC 探针可以在培养物中检测到 mRNA 分子，并且通过添加多个探针可以提高探针检测的灵敏度，这些探针可以很容易地适应强大的 mRNA 定量。使用 FISH-STIC，我们证明了在培养的成纤维细胞中β-肌动蛋白和γ-肌动蛋白 mRNA 的非重叠分布，并在培养的原代海马神经元中检测到神经元特异性转录本。
