This work is supported by grants from (1) the National Key R&#38;D Program of China (2020YFE0201600); (2) the National Nature Science Foundation (81973877 and 82174408); (3) Shanghai Collaborative Innovation Center of Industrial Transformation of Hospital TCM Preparation; (4) Research Projects within Budget of Shanghai University of Traditional Chinese Medicine (2021LK047).
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See the Table of Materials for details of all materials, reagents, and instruments used in this protocol. Figure 1 shows the overall protocol for RNA FISH; Table 1 contains the composition of all solutions and Table 2 contains the primer sequences used in this protocol.
1. Probe preparation
<ol>
	<li>Identify and acquire the FASTA sequence of a target lncRNA of interest, for example, from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Following the indications on the website, design the ISH probes online19, and review the design algorithm&#39;s suggestions to list the probes to be ordered with Cy3 label.</li>
	<li>Incubate the hybridization buffer at 37 &#176;C for 2 h in advance.</li>
	<li>Dissolve 4 optical density (OD) probe in 160 &#181;L of diethylpyrocarbonate (DEPC)-treated ddH2O at the concentration of 1 mg/mL away from light. 
	NOTE: Optical density (OD) represents the measurement unit of DNA and RNA. Usually, 1 OD unit = 33 &#181;g/mL DNA.</li>
	<li>Prepare 200 &#181;L of the probe mixture for each well (1.2 &#181;L-10 &#181;L of probe, 70 &#181;L of hybridization buffer, make up the volume with DEPC-treated ddH2O to 200 &#181;L), and set up a series of 50 &#181;g/mL, 25 &#181;g/mL, 12.5 &#181;g/mL, and 6 &#181;g/mL. 
	NOTE: The probe concentration needs to be explored experimentally in advance. A high probe concentration will lead to non-specific binding of lncRNAs, while a low probe concentration will lead to insensitive or failed detection of lncRNAs.</li>
	<li>Denature the probe mix at 73 &#176;C for 5 min. 
	NOTE: If the Cy3-labeled DNA probe is double-stranded, denature the probe to single-stranded DNA at 95 &#176;C for 5 min, then quickly cool for 2 min on ice.</li>
</ol>
2. Cell preparation
<ol>
	<li>Seed 50,000 143B cells per well on sterile glass coverslips in a 12-well cell culture plate and incubate them for 24 h (37 &#176;C, 5% CO2) in DMEM. 
	NOTE: The specific cell number seeded here varies with cell size, which is appropriate for the seeded cells to reach a confluence of 50% after being incubated for 24 h. The sterile glass coverslips are round to be suitable for the 12-well plate.</li>
	<li>Remove the medium and wash for 2 x 5 min with 1x phosphate-buffered saline (PBS). 
	NOTE: Prepare 1x PBS with DEPC-treated ddH2O. PBS without DEPC-treatment cannot be used because it contains RNase. Perform all the following steps in RNase-free conditions.</li>
	<li>Remove 1x PBS and add 200 &#181;L of 100% ethanol in each well to fix for 15 min at room temperature.</li>
	<li>Remove the ethanol, add 200 &#181;L of 0.1% Triton X-100 (in 1x PBS) in each well, and incubate for 15 min at room temperature. 
	NOTE: The time needs to be strictly controlled with Triton X-100 permeabilization and cannot be too long.</li>
	<li>Remove 0.1% Triton X-100 and wash 2 x 5 min with 1x PBS. 
	NOTE: If the protocol has to be paused here, replace the PBS with 70% ethanol (dilution of 100% ethanol with RNase-free ddH2O) and store the sample at 4 &#176;C for up to 3 months.</li>
	<li>Remove 1x PBS, add 200 &#181;L of 2x sodium saline citrate (SSC) buffer (dilution of 20x SSC with RNase-free ddH2O) into each well, and incubate for 30 min at 37 &#176;C.</li>
	<li>Remove 2x SSC buffer, add 200 &#181;L of 70% ethanol into each well, and incubate for 3 min at room temperature.</li>
	<li>Discard the 70% ethanol, add 200 &#181;L of 85% ethanol (dilution of 100% ethanol with RNase-free ddH2O) into each well, and incubate for 3 min at room temperature.</li>
	<li>Discard the 85% ethanol, add 200 &#181;L of 100% ethanol into each well, and incubate for 3 min at room temperature.</li>
	<li>Absorb and discard 100% ethanol; let the wells dry.</li>
</ol>
3. Fluorescence in situ hybridization (FISH)
<ol>
	<li>Add 200 &#181;L of probe mixture (denatured as in step 1.5) to each well and incubate at 37 &#176;C overnight (16-18 h). 
	NOTE: There is a strong positive correlation between hybridization temperature and probe concentration; therefore, to optimize the background, both the hybridization temperature and the probe concentration should be reduced.</li>
	<li>The next day, take out samples from 37 &#176;C and discard the probe mixture. Add 200 &#181;L of 0.4x SSC/0.3% Tween-20 buffer to each well (preheated at 65 &#176;C) and wash for 2 min at room temperature.</li>
	<li>Remove the 0.4x SSC/0.3% Tween-20 buffer, add 200 &#181;L of 2x SSC/0.1% Tween-20 buffer to each well, and wash for 2 min at room temperature.</li>
	<li>Remove 2x SSC/0.1% Tween-20 buffer, add 200 &#181;L of 4&#39;,6-diamidino-2-phenylindole (DAPI) staining solution (1 &#181;g/mL), and stain for 20 min away from light.</li>
	<li>Discard the DAPI dye solution and wash with 1x PBS for 2 min at room temperature.</li>
	<li>Add 50 &#181;L of mounting medium containing gum onto the slide and place the glass coverslip on the slide to fix. 
	NOTE: Be sure to place the coverslip on the slide with the cell side down.</li>
	<li>Observe under a fluorescence microscope. 
	NOTE: Use a fluorescence microscope or a laser confocal microscope; the latter produces a higher sensitivity and clarity imaging.</li>
</ol>

FISH for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

<ol><li>Djebali, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Landscape+of+transcription+in+human+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Insight+into+the+role+of+long+noncoding+RNA+in+cancer+development+and+progression%5BTitle%5D)+AND+%22International+Review+of+Cell+and+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((lncRNA+FBXL19-AS1+regulates+osteosarcoma+cell+proliferation%2C+migration+and+invasion+by+sponging+miR-346%5BTitle%5D)+AND+%22OncoTargets+and+Therapy%22%5BJournal%5D)">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/pubmed?term=((lncRNA+C2dat1+promotes+cell+proliferation%2C+migration%2C+and+invasion+by+targeting+miR-34a-5p+in+osteosarcoma+cells%5BTitle%5D)+AND+%22Oncology+Research%22%5BJournal%5D)">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/pubmed?term=((LncRNA+MATN1-AS1+for+prediction+of+prognosis+in+osteosarcoma+patients+and+its+cellular+function%5BTitle%5D)+AND+%22Molecular+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Methods+to+study+long+noncoding+RNA+biology+in+cancer%5BTitle%5D)+AND+%22Advances+in+Experimental+Medicine+and+Biology%22%5BJournal%5D)">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/pubmed?term=((lncRNA+TUSC7+inhibits+osteosarcoma+progression+through+the+miR-181a%2FRASSF6+axis%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Medicine%22%5BJournal%5D)">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/pubmed?term=((LncRNA+RUSC1-AS1+promotes+osteosarcoma+progression+through+regulating+the+miR-340-5p+and+PI3K%2FAKT+pathway%5BTitle%5D)+AND+%22Aging%22%5BJournal%5D)">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/pubmed?term=((ceRNA+in+cancer%3A+possible+functions+and+clinical+implications%5BTitle%5D)+AND+%22Journal+of+Medical+Genetics%22%5BJournal%5D)">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/pubmed?term=((RNA+fluorescence+in+situ+hybridization+in+cultured+mammalian+cells%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Visualization+of+single+RNA+transcripts+in+situ%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Dramatically+improved+RNA+in+situ+hybridization+signals+using+LNA-modified+probes%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">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/pubmed?term=((Single-copy+gene+detection+using+branched+DNA+%28bDNA%29+in+situ+hybridization%5BTitle%5D)+AND+%22The+Journal+of+Histochemistry+and+Cytochemistry%22%5BJournal%5D)">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/pubmed?term=((Simultaneous+visualization+of+RNA+transcripts+and+proteins+in+whole-mount+mouse+preimplantation+embryos+using+single-molecule+fluorescence+in+situ+hybridization+and+immunofluorescence+microscopy%5BTitle%5D)+AND+%22Frontiers+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((lncRNA+SNHG6+regulates+EZH2+expression+by+sponging+miR-26a%2Fb+and+miR-214+in+colorectal+cancer%5BTitle%5D)+AND+%22Journal+of+Hematology+%26+Oncology%22%5BJournal%5D)">lncRNA SNHG6 regulates EZH2 expression by sponging miR-26a/b and miR-214 in colorectal cancer.</a> Journal of Hematology & 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/pubmed?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%5BTitle%5D)+AND+%22Cellular+Physiology+and+Biochemistry%3A+International+Journal+of+Experimental+Cellular+Physiology%2C+Biochemistry%2C+and+Pharmacology%22%5BJournal%5D)">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/pubmed?term=((Silencing+of+SNHG6+induced+cell+autophagy+by+targeting+miR-26a-5p%2FULK1+signaling+pathway+in+human+osteosarcoma%5BTitle%5D)+AND+%22Cancer+Cell+International%22%5BJournal%5D)">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/pubmed?term=((Long+non-coding+RNA+SNHG6+as+a+potential+biomarker+for+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Pathology+Oncology+Research%3A+POR%22%5BJournal%5D)">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/pubmed?term=((Detection+of+lncRNA+by+LNA-based+in+situ+hybridization+in+paraffin-embedded+cancer+cell+spheroids%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Long+noncoding+RNA+SNHG6+regulates+p21+expression+via+activation+of+the+JNK+pathway+and+regulation+of+EZH2+in+gastric+cancer+cells%5BTitle%5D)+AND+%22Life+Sciences%22%5BJournal%5D)">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/pubmed?term=((In+situ+hybridization+techniques+for+paraffin-embedded+adult+coral+samples%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">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/pubmed?term=((Single+molecule+RNA+FISH+%28smFISH%29+in+whole-mount+mouse+embryonic+organs%5BTitle%5D)+AND+%22Current+Protocols+in+Cell+Biology%22%5BJournal%5D)">Single molecule RNA FISH (smFISH) in whole-mount mouse embryonic organs.</a> Current Protocols in Cell Biology. 83 (1), 79(2019).</li>
</ol>

The authors declare that they have no competing financial interests.

This RNA FISH protocol can not only detect the localization of lncRNAs in cells, but also detect the colocalization of other RNAs, DNA, or proteins in cells, which can also be used to detect the location of lncRNAs in paraffin-embedded tissues. However, the specific protocol in such cases is different because the paraffin-embedded tissues need to be dewaxed21. This experimental procedure can be applied in 48- or 96-well plates, but 384-well plates are too small to be used here.
<p class="jove_...

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.

<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

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.

Representative images of SNHG6 FISH in human osteosarcoma cells are shown (Figure 2). The negative control is treated with the negative Ctrl probe; positive control is treated with U6 probe 20. SNHG6 probe and U6 probe are labeled with Cy3, which emits red fluorescence. DAPI is a dye that stains the DNA, which emits blue fluorescence. This result shows that SNHG6 is mainly localized in the cytoplasm, and this information can provide an important direction for further ...

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

Author Spotlight: RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells 

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

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

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

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1.4K Views.  Shanghai University of Traditional Chinese Medicine. The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells. 

Video: Author Spotlight: RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells 

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.

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

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.

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

Genetics

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.

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.

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

Biology

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.

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

Neuroscience

Protocol to Prepare Demineralized FFPE Bone Tissue Samples with RNA Integrity 

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.

Author Spotlight: Exploring Advanced Therapeutic Targets in Osteosarcoma Through Spatial Transcriptomics

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.

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

Developmental Biology

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.

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.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

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

Summary

Explore More Videos

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

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

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

Visualizing RNA Localization in Xenopus Oocytes

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

Methods to Enable Spatial Transcriptomics of Bone Tissues

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

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay