这项工作得到了（1）中国国家重点研发计划（2020YFE0201600）的资助;（2）国家自然科学基金（81973877 82174408）;（3）上海市医院中药制剂产业转化协同创新中心;（4）上海中医药大学预算内研究项目（2021LK047）。

有关本协议中使用的所有材料、试剂和仪器的详细信息，请参阅 材料表 。 图1 显示了RNA FISH的整体方案;表 1 包含所有溶液的组成， 表2 包含该协议中使用的引物序列。
1. 探针准备
<ol><li>识别并获取目标靶标 lncRNA 的 FASTA 序列，例如从 GenBank （https://www.ncbi.nlm.nih.gov/genbank/）。按照网站上的指示，在线设计 ISH 探针19，并查看设计算法的建议，以列出要订购的带有 Cy3 标签的探针。</li>
	<li>提前将杂交缓冲液在37°C孵育2小时。</li>
	<li>将 4 个光密度 （OD） 探针溶解在 160 μL 焦碳酸二乙酯 （DEPC） 处理的 ddH2O 中，浓度为 1 mg/mL，远离光。 注：光密度（OD）代表DNA和RNA的测量单位。通常，1 OD 单位 = 33 μg/mL DNA。</li>
	<li>为每个孔准备200μL探针混合物（1.2μL-10μL探针，70μL杂交缓冲液，用DEPC处理的ddH 2O至200μL补足体积），并建立50μg/ mL，25μg/ mL，12.5μg/ mL和6μg/ mL的系列。 注意：探针浓度需要提前通过实验进行探索。高探针浓度会导致lncRNA的非特异性结合，而低探针浓度将导致lncRNA检测不灵敏或检测失败。</li>
	<li>使探针混合物在73°C下变性5分钟。 注：如果Cy3标记的DNA探针是双链的，则在95°C下将探针变性为单链DNA，持续5分钟，然后在冰上快速冷却2分钟。</li>
</ol>2. 细胞制备
<ol><li>在12孔细胞培养板中的无菌玻璃盖玻片上每孔接种50,000个143B细胞，并在DMEM中孵育24小时（37°C，5%CO2）。 注：此处接种的特定细胞数随细胞大小而变化，这适合于接种的细胞在孵育 24 小时后达到 50% 的汇合度。无菌玻璃盖玻片是圆形的，适用于 12 孔板。</li>
	<li>取出培养基，用1x磷酸盐缓冲盐水（PBS）洗涤2 x 5分钟。 注意：用DEPC处理的ddH2O准备1x PBS。在无RNase的条件下执行以下所有步骤。</li>
	<li>取出1x PBS，并在每个孔中加入200μL的100%乙醇，在室温下固定15分钟。</li>
	<li>除去乙醇，在每个孔中加入200μL0.1%Triton X-100（在1x PBS中），并在室温下孵育15分钟。 注意：时间需要用Triton X-100透化严格控制，不能太长。</li>
	<li>除去0.1%Triton X-100，并用1x PBS洗涤2 x 5分钟。 注意：如果必须在此处暂停方案，请用70%乙醇替换PBS（用无RNase的ddH2O稀释100%乙醇），并将样品在4°C下储存长达3个月。</li>
	<li>取出1x PBS，向每个孔中加入200μL2x盐水柠檬酸钠（SSC）缓冲液（用无RNase的ddH2O稀释20x SSC），并在37°C下孵育30分钟。</li>
	<li>取出2x SSC缓冲液，向每个孔中加入200μL 70%乙醇，并在室温下孵育3分钟。</li>
	<li>弃去70%乙醇，向每个孔中加入200μL85%乙醇（用不含RNase的ddH2O稀释100%乙醇），并在室温下孵育3分钟。</li>
	<li>弃去85%乙醇，向每个孔中加入200μL100%乙醇，并在室温下孵育3分钟。</li>
	<li>吸收并丢弃100%乙醇;让井干。</li>
</ol>3. 荧光 原位 杂交（FISH）
<ol><li>向每个孔中加入200μL探针混合物（如步骤1.5所示变性），并在37°C下孵育过夜（16-18小时）。 注：杂交温度与探针浓度之间存在很强的正相关关系;因此，为了优化背景，应降低杂交温度和探针浓度。</li>
	<li>第二天，从37°C取出样品并丢弃探针混合物。向每个孔中加入200μL的0.4x SSC / 0.3%吐温-20缓冲液（在65°C下预热），并在室温下洗涤2分钟。</li>
	<li>取出0.4x SSC / 0.3%吐温-20缓冲液，向每个孔中加入200μL的2x SSC / 0.1%吐温-20缓冲液，并在室温下洗涤2分钟。</li>
	<li>取出 2x SSC/0.1% 吐温-20 缓冲液，加入 200 μL 4'，6-二脒基-2-苯基吲哚 （DAPI） 染色溶液 （1 μg/mL），避光染色 20 分钟。</li>
	<li>弃去DAPI染料溶液，在室温下用1x PBS洗涤2分钟。</li>
	<li>将 50 μL 含有口香糖的封固剂加入载玻片上，并将玻璃盖玻片放在载玻片上进行固定。 注意：务必将盖玻片放在载玻片上，细胞面朝下。</li>
	<li>在荧光显微镜下观察。 注意：使用荧光显微镜或激光共聚焦显微镜;后者产生更高的灵敏度和清晰度成像。</li>
</ol>

FISH 在人骨肉瘤细胞中长链非编码 RNA 定位

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作者声明他们没有相互竞争的经济利益。

该RNA FISH方案不仅可以检测lncRNA在细胞中的定位，还可以检测细胞中其他RNA、DNA或蛋白质的共定位，也可用于检测lncRNA在石蜡包埋组织中的位置。然而，在这种情况下的具体方案是不同的，因为石蜡包埋的组织需要脱蜡21。该实验程序可应用于 48 孔或 96 孔板，但 384 孔板太小，无法在此处使用。
需要注意该方法的几个关键步骤，包括无RNase环境、探针浓度、?...

近年来，全基因组技术的进步极大地扩展了我们对人类基因组的理解。大约93%的人类基因组可以转录成RNA，但只有2%的RNA可以翻译成蛋白质;其余 98% 没有蛋白质翻译功能的 RNA 称为非编码 RNA （ncRNA）1。长链 ncRNA （lncRNA） 是一类非编码 RNA （ncRNA） 中含有 200 多个核苷酸2，由于参与细胞的许多生理和病理过程，如分化、周期调控、凋亡、迁移和侵袭 3,4,5，因此越来越受到关注.LncRNA通过调节染色质结构和核基因表达、控制mRNA剪接过程和转录后修饰等多种机制发挥作用6。LncRNA在转录和转录后水平上调节恶性肿瘤的发生、发展和转移。转录调控是通过与染色体结构结合影响 RNA 转录在细胞核中实现的，而转录后调控是通过内源性竞争性 RNA （ceRNA） 机制控制靶基因在细胞质中实现的 5,7,8。CeRNA揭示了一种新的RNA相互作用机制，即lncRNA可以作为海绵吸附miRNA，抑制miRNA介导的相关靶基因降解9。因此，有关lncRNA的亚细胞定位的信息，无论特定的lncRNA位于细胞质还是细胞核中，对于帮助确定其生物学功能都很重要。
目前，lncRNA定位主要通过两种方法进行检测，一种是通过细胞核/细胞质组分分离试验，另一种是通过RNA FISH。在前者中，分别提取细胞质和细胞核组分中的RNA，然后用特异性lncRNA引物进行PCR扩增，以检测细胞质和细胞核中lncRNA的比例。这种方法的优点是时间效率高，而缺点是实际的lncRNA定位不能直接反映在细胞质和细胞核中lncRNA的相对比例上。RNA FISH可以通过设计lncRNA特异性反义链序列，然后用荧光染料标记来检测细胞中的lncRNA定位10。随着探针技术和检测方法的进步，RNA FISH方法得到了改进，包括荧光团标记的多个寡核苷酸探针组11、LNA探针12和支链DNA（bDNA）探针13。RNA FISH不仅可以检测lncRNA的定位，还可以使用双色或多色免疫荧光14检测其他RNA、DNA或蛋白质的共定位。
在这项工作中，我们以RNA FISH在骨肉瘤细胞（143B）中lncRNA小核仁RNA宿主基因6（SNHG6）的详细细胞内定位检测方案为例。SNHG6 是一种 600-730 核苷酸的成熟剪接形式的 lncRNA，被鉴定为多种人类癌症的新型致癌基因，包括结直肠癌、胃癌、卵巢透明细胞癌、骨肉瘤和肝细胞癌15、16、17、18。研究证实了 SNHG6 参与癌细胞的生物学行为，例如增殖、EMT 和自噬，并显示了 SNHG6 的细胞质定位，它可能通过结合（海绵化）miRNA15、16、17 来影响靶基因。本文介绍了通过RNA FISH进行SNHG6细胞内定位的详细检测方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automatic cell counter</td>
			<td>Shanghai Simo Biological Technology Co., Ltd</td>
			<td>IC1000</td>
			<td>Counting cells</td>
		</tr>
		<tr>
			<td>Cell culture plate-12</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>3513,corning</td>
			<td>Place the coverslips in the plate</td>
		</tr>
		<tr>
			<td>&#160;Cell line (143B)</td>
			<td>Cell Bank of Chinese Academy of Sciences</td>
			<td>CRL-8303</td>
			<td>osteosarcoma cancer cell line</td>
		</tr>
		<tr>
			<td>Centrifuge tube (15 mL, 50 mL)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>&#160;430790, Corning</td>
			<td>Centrifuge the cells</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>abs7026</td>
			<td>The cells are seeded on the coverslips</td>
		</tr>
		<tr>
			<td>Cy3 label-SNHG6 DNA probe</td>
			<td>Shanghai GenePharma Co.,Ltd</td>
			<td>A10005</td>
			<td>Detect SNHG6 location</td>
		</tr>
		<tr>
			<td>DMEM media</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>LM-E1141</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Dry Bath Incubator</td>
			<td>Haimen Kylin-Bell Lab Instruments Co.,Ltd.</td>
			<td>DKT200-2&#160;</td>
			<td>Incubation at different high temperatures</td>
		</tr>
		<tr>
			<td>Ethanol 100%&#160;</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>10009218</td>
			<td>dehydration</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Shanghai Waihai Biotechnology Co., LTD</td>
			<td>Olympus BX43 equipped with a camera of Olympus U-TV0.5XC-3(SN:5J01719),olympus</td>
			<td>Observation and positioning</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Shanghai Yiheng Scientific Instrument Co., LTD</td>
			<td>DHP-9051</td>
			<td>The samples were incubated at 37 &#176;C.</td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>E675004</td>
			<td>Attach the coverslips to the slide</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Haimen Kylin-Bell Lab Instruments Co.,Ltd.</td>
			<td>TS-8S</td>
			<td>Washing sample</td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>188105</td>
			<td>The coverslips is placed on the slide</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A600198</td>
			<td>Permeable membrane and nuclear membrane</td>
		</tr>
		<tr>
			<td>&#160;Trypsin (0.25%)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>25200056, Gibco</td>
			<td>trypsin treatment of cells</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A600560</td>
			<td>detergent</td>
		</tr>
	</tbody>
</table>

rna fluorescence in situ hybridization for long non-coding rna localization in human osteosarcoma cells

已经研究了长链非编码RNA（lncRNA）在癌症中的重要作用，如调节癌细胞的增殖、上皮-间充质转化（EMT）、迁移、浸润和自噬。细胞中lncRNA的定位检测可以深入了解其功能。通过设计lncRNA特异性反义链序列，然后用荧光染料标记，RNA荧光 原位 杂交（FISH）可用于检测lncRNA的细胞定位。随着显微镜的发展，RNA FISH技术现在甚至可以对表达不良的lncRNA进行可视化。该方法不仅可以单独检测lncRNA的定位，还可以利用双色或多色免疫荧光检测其他RNA、DNA或蛋白质的共定位。在此，我们以人骨肉瘤细胞中的lncRNA小核仁RNA宿主基因6（SNHG6）为例，对RNA FISH的详细实验操作步骤和注意事项进行了介绍，以期为想要进行RNA FISH实验，尤其是lncRNA FISH的研究人员提供参考。

Our understanding of the human genome has been greatly expanded by recent advances in whole-genome technology. About 93% of the human genome can be transcribed into RNAs, but only 2% of the RNAs can be translated into proteins; the remaining 98% of RNAs that have no protein translation function are called non-coding RNA (ncRNA)1. As a class of noncoding RNAs (ncRNAs), long ncRNAs (lncRNAs), containing over 200 nucleotides2, have attracted increasing attention due to their involvement in many physiological and pathological processes of the cells, such as differentiation, cycle control, apoptosis, migration, and invasion3,4,5. LncRNAs play their roles through various mechanisms, such as regulating chromatin structure and nuclear gene expression, controlling the mRNA splicing process, and posttranscriptional modification6. LncRNAs regulate the occurrence, development, and metastasis of malignancies at both the transcriptional and posttranscriptional levels. Transcriptional regulation is realized in the nucleus by affecting RNA transcription via binding to the chromosomal structures, while posttranscriptional regulation is realized in the cytoplasm by controlling the target genes via an endogenous competitive RNA (ceRNA) mechanism5, 7, 8. CeRNA has revealed a new mechanism of RNA interaction, namely that lncRNAs can act as a sponge to adsorb miRNAs and inhibit the miRNA-mediated degradation of related target genes9. Therefore, the information regarding the subcellular localization of lncRNAs, whether a specific lncRNA is located in the cytoplasm or nucleus, is important to help identify their biological functions.
At present, lncRNA localization is mainly detected by two methods, one is by nucleus/cytoplasm fraction isolation assay, and the other is by RNA FISH. In the former, RNAs in the cytoplasmic and the nuclear fractions are extracted respectively, and then PCR amplification is performed with specific lncRNA primers to detect the ratio of lncRNAs in the cytoplasm and nucleus. The advantage of this method is time efficiency, while the disadvantage is that the actual lncRNA localization is not directly reflected by the relative proportion of lncRNAs in the cytoplasm and nucleus. RNA FISH can detect lncRNA localization in cells through the design of lncRNA-specific antisense chain sequences followed by labeling with fluorescent dyes10. The RNA FISH methods have been improved with advances in probe techniques and detection methods, including fluorophore-labeled multiple oligo probe sets11, LNA probes12, and branched-DNA (bDNA) probes13. RNA FISH can not only detect the localization of lncRNA, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence14.
In this work, we have included the detailed intracellular localization detection protocol of lncRNA small nucleolar RNA host gene 6 (SNHG6) in osteosarcoma cells (143B) by RNA FISH as an example. SNHG6 is a 600-730 nucleotide lncRNA in its mature spliced form and identified as a novel oncogene in diverse human cancers, including colorectal cancer, gastric cancer, ovarian clear cell carcinoma, osteosarcoma, and hepatocellular carcinoma15,16,17,18. Studies have confirmed the involvement of SNHG6 in biological behaviors of cancer cells, such as proliferation, EMT, and autophagy, and shown the cytoplasmic localization of SNHG6 where it may affect the target genes by binding (sponging) the miRNAs15,16,17. This detailed detection protocol of SNHG6 intracellular localization by RNA FISH is presented herein.

作者聚焦：用于在骨肉瘤细胞中定位 lncRNA-SNHG6 的 RNA FISH 

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

The scope of our research is to provide a detailed RNA FISH protocol that's providing a reference for scientists who want to perform RNA FISH experiments. Our research has established that RNA FISH can be used to localize lncRNA in human osteosarcoma cells. Our protocol provided a comprehensive experimental procedure and the precautions for RNA FISH using lncRNA-SNHG6 in human osteosarcoma cells as a reference.

显示了人骨肉瘤细胞中SNHG6 FISH的代表性图像（图2）。阴性对照用阴性Ctrl探针处理;阳性对照用U6探针 20处理。SNHG6探针和U6探针用Cy3标记，Cy3发出红色荧光。DAPI是一种染料，可对DNA进行染色，从而发出蓝色荧光。这一结果表明，SNHG6主要定位于细胞质中，这些信息可为SNHG6的进一步研究提供重要方向。
如果使用非常高浓度的探针，则可?...

Watch this Scientific Journal Video about RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells at JoVE.com

本方案描述了一种RNA荧光 原位 杂交以在人骨肉瘤细胞中定位lncRNA的方法。

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition ...

我们的研究范围是提供详细的RNA FISH方案，为想要进行RNA FISH实验的科学家提供参考。我们的研究已经确定，RNA FISH可用于定位人骨肉瘤细胞中的lncRNA。我们的实验方案以lncRNA-SNHG6为参考，提供了全面的实验程序和RNA FISH在人骨肉瘤细胞中的注意事项。

rna-fluorescence-situ-hybridization-for-long-non-coding-rna

Research

JoVE Journal

Cancer Research

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

1.0K 次观看.  Shanghai University of Traditional Chinese Medicine. 我们的研究范围是提供详细的RNA FISH方案，为想要进行RNA FISH实验的科学家提供参考。我们的研究已经确定，RNA FISH可用于定位人骨肉瘤细胞中的lncRNA。我们的实验方案以lncRNA-SNHG6为参考，提供了全面的实验程序和RNA FISH在人骨肉瘤细胞中的注意事项。

视频: 作者聚焦：用于在骨肉瘤细胞中定位 lncRNA-SNHG6 的 RNA FISH 

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells.

科研

癌症研究

RNA Fluorescence In Situ Hybridization to Localize the lncRNAs in Human Osteosarcoma Cells

To begin, after acquiring the long non-coding RNA of interest, incubate the hybridization buffer at 37 degrees Celsius for two hours. Next, dissolve the four optical density probe in 160 microliters of diethyl pyrocarbonate treated deionized water away from the light. Prepare 200 microliters of the probe mixture for each well, and set up a series of 50, 25, 12.5, and six micrograms per milliliter.Denature the probe mix at 73 degrees Celsius for five minutes. Take a 12-well cell culture plate containing 143B cells on sterile glass cover slips. Remove the medium and wash twice for five minutes with PBS.Place the cells on a shaker. Then remove the PBS and add 200 microliters of 100%ethanol to each well, fixing for 15 minutes at room temperature. Then remove the ethanol, add 200 microliters of 0.1%Triton X-100 to each well, and incubate for 15 minutes at room temperature.Remove 0.1%Triton X-100 and wash two times for five minutes with PBS. Next, remove PBS, add 200 microliters of two times sodium-saline citrate or SSC buffer into each well, and incubate for 30 minutes at 37 degrees Celsius. Remove two times SSC buffer, add 200 microliters of 70%ethanol into each well, and incubate for three minutes at room temperature.Discard the 70%ethanol, then add 200 microliters of 85%ethanol into each well and incubate for three minutes at room temperature. Next, discard the 85%ethanol, add 200 microliters of 100%ethanol to each well, and incubate for three minutes at room temperature. Afterward, absorb and discard the 100%ethanol and allow the wells to dry.Next, add 200 microliters of denatured probe mixture to each well, then incubate at 37 degrees Celsius overnight. The following day, remove samples from 37 degrees Celsius, discard the probe mixture, and add 200 microliters of preheated 0.4 times SSC 0.3%Tween 20 buffer to each well and wash for two minutes at room temperature. Remove the 0.4 times SSC 0.3%Tween 20 buffer.Add 200 microliters of two times SSC 0.1%Tween 20 buffer to each well and wash for two minutes at room temperature. Next, discard the two times SSC 0.1%Tween 20 buffer. Add 200 microliters of DAPI staining solution and stain for 20 minutes in the dark on a shaker.After staining, discard the DAPI solution and wash it with PBS for two minutes at room temperature. Lastly, add 50 microliters of mounting medium containing gum to the slide and place the glass cover slip to fix the slide. Representative SNHG6 FISH images in human osteosarcoma cells are shown.SNHG6 probe labeled with Cy3, which emits red fluorescence, showed that SNHG6 is mainly localized in the cytoplasm. A background color was observed when using a high concentration of probe.