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

<ol>
	<li>Djebali, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landscape+of+transcription+in+human+cells.">Landscape of transcription in human cells.</a> Nature. 489 (7414), 101-108 (2012).</li><li>Li, C. H., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+role+of+long+noncoding+RNA+in+cancer+development+and+progression.">Insight into the role of long noncoding RNA in cancer development and progression.</a> International Review of Cell and Molecular Biology. 326, 33-65 (2016).</li><li>Pan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=lncRNA+FBXL19-AS1+regulates+osteosarcoma+cell+proliferation,+migration+and+invasion+by+sponging+miR-346.">lncRNA FBXL19-AS1 regulates osteosarcoma cell proliferation, migration and invasion by sponging miR-346.</a> OncoTargets and Therapy. 11, 8409-8420 (2018).</li><li>Jia, D., Niu, Y., Li, D., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=lncRNA+C2dat1+promotes+cell+proliferation,+migration,+and+invasion+by+targeting+miR-34a-5p+in+osteosarcoma+cells.">lncRNA C2dat1 promotes cell proliferation, migration, and invasion by targeting miR-34a-5p in osteosarcoma cells.</a> Oncology Research. 26 (5), 753-764 (2018).</li><li>Liu, Y., Wang, D., Ji, Q., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LncRNA+MATN1-AS1+for+prediction+of+prognosis+in+osteosarcoma+patients+and+its+cellular+function.">LncRNA MATN1-AS1 for prediction of prognosis in osteosarcoma patients and its cellular function.</a> Molecular Biotechnology. 64 (1), 66-74 (2022).</li><li>Luo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+study+long+noncoding+RNA+biology+in+cancer.">Methods to study long noncoding RNA biology in cancer.</a> Advances in Experimental Medicine and Biology. 927, 69-107 (2016).</li><li>Zhao, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=lncRNA+TUSC7+inhibits+osteosarcoma+progression+through+the+miR-181a/RASSF6+axis.">lncRNA TUSC7 inhibits osteosarcoma progression through the miR-181a/RASSF6 axis.</a> International Journal of Molecular Medicine. 47 (2), 583-594 (2021).</li><li>Tong, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LncRNA+RUSC1-AS1+promotes+osteosarcoma+progression+through+regulating+the+miR-340-5p+and+PI3K/AKT+pathway.">LncRNA RUSC1-AS1 promotes osteosarcoma progression through regulating the miR-340-5p and PI3K/AKT pathway.</a> Aging. 13 (16), 20116-20130 (2021).</li><li>Qi, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ceRNA+in+cancer:+possible+functions+and+clinical+implications.">ceRNA in cancer: possible functions and clinical implications.</a> Journal of Medical Genetics. 52 (10), 710-718 (2015).</li><li>Tripathi, V., Fei, J., Ha, T., Prasanth, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+fluorescence+in+situ+hybridization+in+cultured+mammalian+cells.">RNA fluorescence in situ hybridization in cultured mammalian cells.</a> Methods in Molecular Biology. 1206, 123-136 (2015).</li><li>Femino, A. M., Fay, F. S., Fogarty, K., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+single+RNA+transcripts+in+situ.">Visualization of single RNA transcripts in situ.</a> Science. 280 (5363), 585-590 (1998).</li><li>Thomsen, R., Nielsen, P. S., Jensen, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatically+improved+RNA+in+situ+hybridization+signals+using+LNA-modified+probes.">Dramatically improved RNA in situ hybridization signals using LNA-modified probes.</a> RNA. 11 (11), 1745-1748 (2005).</li><li>Player, A. N., Shen, L. P., Kenny, D., Antao, V. P., Kolberg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-copy+gene+detection+using+branched+DNA+(bDNA)+in+situ+hybridization.">Single-copy gene detection using branched DNA (bDNA) in situ hybridization.</a> The Journal of Histochemistry and Cytochemistry. 49 (5), 603-612 (2001).</li><li>Hazra, R., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+visualization+of+RNA+transcripts+and+proteins+in+whole-mount+mouse+preimplantation+embryos+using+single-molecule+fluorescence+in+situ+hybridization+and+immunofluorescence+microscopy.">Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy.</a> Frontiers in Cell and Developmental Biology. 10, 986261 (2022).</li><li>Xu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=lncRNA+SNHG6+regulates+EZH2+expression+by+sponging+miR-26a/b+and+miR-214+in+colorectal+cancer.">lncRNA SNHG6 regulates EZH2 expression by sponging miR-26a/b and miR-214 in colorectal cancer.</a> Journal of Hematology &amp; Oncology. 12 (1), 3 (2019).</li><li>Yan, K., Tian, J., Shi, W., Xia, H., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LncRNA+SNHG6+is+associated+with+poor+prognosis+of+gastric+cancer+and+promotes+cell+proliferation+and+EMT+through+epigenetically+silencing+p27+and+sponging+miR-101-3p.">LncRNA SNHG6 is associated with poor prognosis of gastric cancer and promotes cell proliferation and EMT through epigenetically silencing p27 and sponging miR-101-3p.</a> Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology. 42 (3), 999-1012 (2017).</li><li>Zhu, X., Yang, G., Xu, J., Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+SNHG6+induced+cell+autophagy+by+targeting+miR-26a-5p/ULK1+signaling+pathway+in+human+osteosarcoma.">Silencing of SNHG6 induced cell autophagy by targeting miR-26a-5p/ULK1 signaling pathway in human osteosarcoma.</a> Cancer Cell International. 19, 82 (2019).</li><li>Birgani, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA+SNHG6+as+a+potential+biomarker+for+hepatocellular+carcinoma.">Long non-coding RNA SNHG6 as a potential biomarker for hepatocellular carcinoma.</a> Pathology Oncology Research: POR. 24 (2), 329-337 (2018).</li><li>Nielsen, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+lncRNA+by+LNA-based+in+situ+hybridization+in+paraffin-embedded+cancer+cell+spheroids.">Detection of lncRNA by LNA-based in situ hybridization in paraffin-embedded cancer cell spheroids.</a> Methods in Molecular Biology. 2348, 123-137 (2021).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+noncoding+RNA+SNHG6+regulates+p21+expression+via+activation+of+the+JNK+pathway+and+regulation+of+EZH2+in+gastric+cancer+cells.">Long noncoding RNA SNHG6 regulates p21 expression via activation of the JNK pathway and regulation of EZH2 in gastric cancer cells.</a> Life Sciences. 208, 295-304 (2018).</li><li>Traylor-Knowles, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+techniques+for+paraffin-embedded+adult+coral+samples.">In situ hybridization techniques for paraffin-embedded adult coral samples.</a> Journal of Visualized Experiments: JoVE. (138), e57853 (2018).</li><li>Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+RNA+FISH+(smFISH)+in+whole-mount+mouse+embryonic+organs.">Single molecule RNA FISH (smFISH) in whole-mount mouse embryonic organs.</a> Current Protocols in Cell Biology. 83 (1), 79 (2019).</li></ol>

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><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>&amp;nbsp;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>&amp;nbsp;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&amp;nbsp;</td><td>Incubation at different high temperatures</td></tr><tr><td>Ethanol 100%&amp;nbsp;</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 &amp;deg;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>&amp;nbsp;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。

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

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

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。

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

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

1.3K 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 

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

巨蟹座的建立源于人类传统的骨肉瘤细胞培养

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

科研

癌症研究

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

遗传学

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

生物学

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

神经科学

制备具有 RNA 完整性的脱矿质 FFPE 骨组织样品的方案 

Methods to Enable Spatial Transcriptomics of Bone Tissues

Understanding the relationship between the cells and their location within each tissue is critical to uncover the biological processes associated with normal development and disease pathology. Spatial transcriptomics is a powerful method that enables the analysis of the whole transcriptome within tissue samples, thus providing information about the cellular gene expression and the histological context in which the cells reside. While this method has been extensively utilized for many soft tissues, its application for the analyses of hard tissues such as bone has been challenging. The major challenge resides in the inability to preserve good quality RNA and tissue morphology while processing the hard tissue samples for sectioning. Therefore, a method is described here to process freshly obtained bone tissue samples to effectively generate spatial transcriptomics data. The method allows for the decalcification of the samples, granting successful tissue sections with preserved morphological details while avoiding RNA degradation. In addition, detailed guidelines are provided for samples that were previously paraffin-embedded, without demineralization, such as&#160;samples collected from tissue banks. Using these guidelines, high-quality spatial transcriptomics data generated from tissue bank samples of primary tumor and lung metastasis of bone osteosarcoma are shown.

作者聚焦:通过空间转录组学探索骨肉瘤的先进治疗靶点

Here, we describe a method that allows for the decalcification of freshly obtained bone tissues and the preservation of high-quality RNA. A method is also illustrated for sectioning Formalin Fixed Paraffin Embedded (FFPE) samples of non-demineralized bones to obtain good quality results if fresh tissues are not available or cannot be collected.

实现骨组织空间转录组学的方法

Understanding the relationship between the cells and their location within each tissue is critical to uncover the biological processes associated with ...

发育生物学

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

副本

摘要

探索更多视频

此视频中的章节

巨蟹座的建立源于人类传统的骨肉瘤细胞培养

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率

可视化的RNA中的本地化爪蟾卵母细胞

在脑切片检测轴突本地化mRNAs的高空间分辨率

实现骨组织空间转录组学的方法

实现骨组织空间转录组学的方法

实现骨组织空间转录组学的方法

放射性的原位杂交检测组织不同的基因表达模式

全山RNA荧光

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

副本

摘要

探索更多视频

此视频中的章节

巨蟹座的建立源于人类传统的骨肉瘤细胞培养

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率

可视化的RNA中的本地化爪蟾卵母细胞

在脑切片检测轴突本地化mRNAs的高空间分辨率

实现骨组织空间转录组学的方法

实现骨组织空间转录组学的方法

实现骨组织空间转录组学的方法

放射性的原位杂交检测组织不同的基因表达模式

全山RNA荧光

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位