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).
&#160;&#160;&#160;&#160;

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/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>

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 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

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 ...

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

Research

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Cancer Research

1.2K 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 

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education.
Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2.
Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2.

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled.
These authors&#39; laboratories&#39; contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue.

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...

Diagnosis of Vitreoretinal Lymphoma Using Cell-Free DNA

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Author Spotlight: Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor 

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To ...

Protocol for Osteosarcoma PDX Mouse Models: Studying Growth and Drug Resistance

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

Author Spotlight: Replicating Human Osteosarcoma Progression in Immunodeficient Mice for Cancer Study

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...