ai_agent

この研究は、(1)中国国家重点研究開発プログラム(2020YFE0201600)からの助成金によって支援されています。(2)国立自然科学財団(81973877および82174408)。(3)病院TCM準備の産業変革の上海共同イノベーションセンター。(4)上海中医薬大学予算内の研究プロジェクト(2021LK047)。

このプロトコルで使用されるすべての材料、試薬、および機器の詳細については、 材料表 を参照してください。 図1 は、RNA FISHの全体的なプロトコルを示しています。表 1 にはすべての溶液の組成が含まれ、 表2 にはこのプロトコルで使用されるプライマー配列が含まれています。
1. プローブの準備
<ol><li>例えば、GenBank(https://www.ncbi.nlm.nih.gov/genbank/)から、目的の標的lncRNAのFASTA配列を同定し、取得する。ウェブサイトの指示に従って、ISHプローブをオンラインで設計し19、設計アルゴリズムの提案を確認して、Cy3ラベルで注文するプローブをリストします。</li>
	<li>ハイブリダイゼーションバッファーを事前に37°Cで2時間インキュベートします。</li>
	<li>4光学密度(OD)プローブを160 μLのジエチルピロカーボネート(DEPC)処理ddH2Oに、光から1 mg/mLの濃度で溶解します。 注:光学濃度(OD)は、DNAおよびRNAの測定単位を表します。通常、1 OD 単位 = 33 μg/mL の DNA。</li>
	<li>各ウェルについて200μLのプローブ混合物(1.2μL〜10μLのプローブ、70μLのハイブリダイゼーションバッファー、DEPC処理ddH2O〜200μLで容量を構成します)を調製し、50μg/mL、25μg/mL、12.5μg/mL、および6μg/mLのシリーズをセットアップします。 注:プローブの濃度は、事前に実験的に調査する必要があります。プローブ濃度が高いとlncRNAが非特異的に結合し、プローブ濃度が低いとlncRNAの感度が低下したり、検出に失敗したりします。</li>
	<li>プローブミックスを73°Cで5分間変性させます。 注:Cy3標識DNAプローブが二本鎖の場合は、プローブを95°Cで5分間一本鎖DNAに変性させた後、氷上で2分間急速に冷却します。</li>
</ol>2. 細胞調製
<ol><li>12ウェル細胞培養プレート内の滅菌ガラスカバーガラス上にウェルあたり50,000個の143B細胞を播種し、DMEM中で24時間(37°C、5%CO2)インキュベートします。 注:ここで播種する特定の細胞数は細胞サイズによって異なり、播種した細胞が24時間インキュベートした後に50%の合流に達するのに適しています。滅菌ガラスカバーガラスは丸型で、12ウェルプレートに適しています。</li>
	<li>培地を取り出し、1xリン酸緩衝生理食塩水(PBS)で2 x 5分間洗浄します。 注:DEPC処理されたddH2OのPBSを1x 調製します。以下のすべての手順を RNase フリーの条件で実行します。</li>
	<li>1x PBSを除去し、各ウェルに200 μLの100%エタノールを加えて、室温で15分間固定します。</li>
	<li>エタノールを除去し、各ウェルに 200 μL の 0.1% Triton X-100(1x PBS 中)を加え、室温で 15 分間インキュベートします。 注:時間はTriton X-100透過処理で厳密に制御する必要があり、長すぎることはできません。</li>
	<li>0.1% Triton X-100を除去し、1x PBSで5分×2回洗浄します。 注:ここでプロトコルを一時停止する必要がある場合は、PBS を 70% エタノール(100% エタノールを RNase フリー ddH2O で希釈)に交換し、サンプルを 4 °C で最大 3 か月間保存します。</li>
	<li>1x PBSを除去し、2x生理食塩水クエン酸ナトリウム(SSC)バッファー200 μL(RNaseフリーddH2Oで20x SSCを希釈)を各ウェルに加え、37°Cで30分間インキュベートします。</li>
	<li>2x SSCバッファーを除去し、各ウェルに200 μLの70%エタノールを加え、室温で3分間インキュベートします。</li>
	<li>70%エタノールを廃棄し、200μLの85%エタノール(100%エタノールをRNaseフリーddH2Oで希釈)を各ウェルに加え、室温で3分間インキュベートします。</li>
	<li>85%エタノールを廃棄し、各ウェルに100%エタノール200μLを加え、室温で3分間インキュベートします。</li>
	<li>100%エタノールを吸収して廃棄します。井戸を乾かします。</li>
</ol>3. 蛍光 in situ ハイブリダイゼーション(FISH)
<ol><li>200μLのプローブ混合物(ステップ1.5と同様に変性)を各ウェルに加え、37°Cで一晩(16〜18時間)インキュベートします。 注:ハイブリダイゼーション温度とプローブ濃度の間には強い正の相関関係があります。したがって、バックグラウンドを最適化するには、ハイブリダイゼーション温度とプローブ濃度の両方を下げる必要があります。</li>
	<li>翌日、37°Cからサンプルを取り出し、プローブ混合物を廃棄します。200 μL の 0.4x SSC/0.3% Tween-20 バッファーを各ウェル(65 °C に予熱)に加え、室温で 2 分間洗浄します。</li>
	<li>0.4x SSC/0.3% Tween-20 バッファーを除去し、2x SSC/0.1% Tween-20 バッファー 200 μL を各ウェルに加え、室温で 2 分間洗浄します。</li>
	<li>2x SSC/0.1% Tween-20バッファーを除去し、200 μLの4',6-ジアミジノ-2-フェニルインドール(DAPI)染色溶液(1 μg/mL)を加え、光を避けて20分間染色します。</li>
	<li>DAPI色素溶液を廃棄し、1x PBSで室温で2分間洗浄します。</li>
	<li>ガムを含む封入剤50 μLをスライドに加え、ガラスカバーガラスをスライドの上に置いて固定します。 注意: カバーガラスは、必ずセル側を下にしてスライドに置いてください。</li>
	<li>蛍光顕微鏡で観察します。 注意: 蛍光顕微鏡またはレーザー共焦点顕微鏡を使用してください。後者は、より高い感度と明瞭なイメージングを生成します。</li>
</ol>

ヒト骨肉腫細胞における長いノンコーディングRNA局在のためのFISH

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<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>

著者らは、競合する金銭的利害関係はないと宣言しています。

このRNA FISHプロトコルは、セル内のlncRNAの局在を検出するだけでなく、他のRNA、DNA、または細胞内のタンパク質の共局在を検出することもでき、パラフィン包埋組織におけるlncRNAの位置を検出するために使用できます。しかし、そのような場合の特定のプロトコルは、パラフィン包埋組織を脱ワックスする必要があるため、異なる21。この実験手順は、48ウェルまたは96ウェ?...

ヒトゲノムの理解は、近年の全ゲノム技術の進歩によって大きく拡大しています。ヒトゲノムの約93%はRNAに転写できますが、タンパク質に翻訳できるRNAはわずか2%です。タンパク質翻訳機能を持たないRNAの残りの98%は、ノンコーディングRNA(ncRNA)1と呼ばれます。200以上のヌクレオチド2を含む長鎖ncRNA(lncRNA)は、分化、周期制御、アポトーシス、遊走、浸潤など、細胞の多くの生理学的・病理学的プロセスに関与していることから、ノンコーディングRNA(ncRNA)のクラスとしてますます注目を集めています3,4,5。.LncRNAは、クロマチン構造や核内遺伝子発現の調節、mRNAスプライシングプロセスの制御、転写後修飾など、さまざまなメカニズムを通じてその役割を果たしています6。LncRNAは、転写レベルと転写後レベルの両方で悪性腫瘍の発生、発生、および転移を調節します。核内では染色体構造への結合を介してRNAの転写に作用することで転写制御が実現され、細胞質内では内因性競合RNA(ceRNA)機構を介して標的遺伝子を制御することで転写後制御が実現される5,7,8。CeRNAは、lncRNAがスポンジとしてmiRNAを吸着し、miRNAを介した関連標的遺伝子の分解を阻害するという、RNA相互作用の新しいメカニズムを明らかにしました9。したがって、lncRNAの細胞内局在に関する情報は、特定のlncRNAが細胞質または核に存在するかどうか、それらの生物学的機能を特定するために重要です。
現在、lncRNAの局在は、主に核/細胞質分離アッセイとRNA FISHの2つの方法で検出されています。前者では、細胞質画分と核分画のRNAをそれぞれ抽出し、特定のlncRNAプライマーでPCR増幅を行い、細胞質と核のlncRNAの比率を検出します。この方法の利点は時間効率ですが、欠点は、実際のlncRNAの局在が細胞質と核内のlncRNAの相対的な割合によって直接反映されないことです。RNA FISHは、lncRNA特異的なアンチセンス鎖配列を設計し、それに続く蛍光色素で標識することにより、細胞内のlncRNA局在を検出することができます10。RNA FISH法は、蛍光色素標識マルチオリゴプローブセット11、LNAプローブ12、分岐DNA(bDNA)プローブ13など、プローブ技術および検出法の進歩により改良されてきました。RNA FISHは、lncRNAの局在を検出するだけでなく、ダブルカラーまたはマルチカラーの免疫蛍光法を使用して、他のRNA、DNA、またはタンパク質の共局在を検出することもできます14。
本研究では、骨肉腫細胞(143B)におけるlncRNA低核小体RNA宿主遺伝子6(SNHG6)の詳細な細胞内局在検出プロトコルをRNA FISHに例に含めた。SNHG6は、成熟したスプライシング型の600〜730ヌクレオチドlncRNAであり、結腸直腸癌、胃癌、卵巣明細胞癌、骨肉腫、肝細胞癌など、さまざまなヒト癌における新規癌遺伝子として同定されています15,16,17,18。研究により、SNHG6が増殖、EMT、オートファジーなどのがん細胞の生物学的挙動に関与していることが確認されており、SNHG6の細胞質局在が示されており、miRNAに結合(スポンジ化)することで標的遺伝子に影響を与える可能性があります15,16,17。RNA FISHによるSNHG6細胞内局在のこの詳細な検出プロトコルを本明細書に提示する。

<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

がん細胞の増殖、上皮間葉転換(EMT)、遊走、浸潤、オートファジーの調節など、がんにおける長鎖ノンコーディングRNA(lncRNA)の重要な役割が研究されています。細胞内のlncRNAの局在検出は、その機能に関する洞察を提供することができます。lncRNA特異的なアンチセンス鎖配列を設計し、その後に蛍光色素で標識することにより、RNA蛍光 in situ ハイブリダイゼーション(FISH)を適用してlncRNAの細胞局在を検出することができます。顕微鏡法の開発とともに、RNA FISH技術により、発現不良のlncRNAの可視化も可能になりました。この方法では、lncRNAの局在を単独で検出できるだけでなく、ダブルカラーまたはマルチカラーの免疫蛍光法を使用して、他のRNA、DNA、またはタンパク質の共局在を検出することもできます。ここでは、ヒト骨肉腫細胞(143B)におけるlncRNA小核小体RNA宿主遺伝子6(SNHG6)を例に、RNA FISH実験、特にlncRNA FISH実験を行いたい研究者の参考となるように、RNA FISHの詳細な実験操作手順と注意点を記載した。

ヒト骨肉腫細胞におけるSNHG6 FISHの代表的な画像を示します(図2)。ネガティブコントロールは、ネガティブCtrlプローブで処理されます。ポジティブコントロールはU6プローブ 20で処理する。SNHG6プローブとU6プローブは、赤色蛍光を発するCy3で標識されています。DAPIは、青色蛍光を発するDNAを染色する染料です。この結果は、SNHG6が主に細胞質に局在し?...

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

著者スポットライト:骨肉腫細胞におけるlncRNA-SNHG6の位置を特定するためのRNA FISH 

本プロトコルは、ヒト骨肉腫細胞のlncRNAを局在化させるRNA蛍光 in situ ハイブリダイゼーションの方法を記述する。

ヒト骨肉腫細胞における長鎖ノンコーディングRNA局在化のためのRNA蛍光 in situ ハイブリダイゼーション

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

私たちの研究の範囲は、RNA FISH実験を行いたい科学者にリファレンスを提供する詳細なRNA FISHプロトコルを提供することです。私たちの研究は、RNA FISHを使用してヒト骨肉腫細胞のlncRNAを局在化できることを確立しました。私たちのプロトコルは、ヒト骨肉腫細胞のlncRNA-SNHG6を基準としてRNA FISHの包括的な実験手順と注意事項を提供しました。

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

2.0K ビュー.  Shanghai University of Traditional Chinese Medicine. 私たちの研究の範囲は、RNA FISH実験を行いたい科学者にリファレンスを提供する詳細なRNA FISHプロトコルを提供することです。私たちの研究は、RNA FISHを使用してヒト骨肉腫細胞のlncRNAを局在化できることを確立しました。私たちのプロトコルは、ヒト骨肉腫細胞のlncRNA-SNHG6を基準としてRNA FISHの包括的な実験手順と注意事項を提供しました。

ビデオ: 著者スポットライト:骨肉腫細胞におけるlncRNA-SNHG6の位置を特定するためのRNA FISH 

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.

Xenopus OocytesにおけるRNA局在の可視化

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

可視化RNAの局在にアフリカツメガエル卵母細胞

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

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.

組織における多様な遺伝子発現パターンを検出するための放射性in situハイブリダイゼーション

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.

放射性その場で</em組織における多様な遺伝子発現パターンを検出するための>ハイブリダイゼーション

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.

ショウジョウバエ胚のホールマウントRNA蛍光in situハイブリダイゼーション

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.

ホールマウントRNAを蛍光その場でハイブリダイゼーションショウジョウバエ胚

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.

低酸素症後のシングルセルレベルでのグローバルRNA合成の解析

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.

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

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

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.

高分解能In Situハイブリダイゼーションを用いた脳切片における軸索局在mRNAの検出

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.

高解像度を使用した脳切片における軸索のローカライズされたmRNAの検出その場でのハイブリダイゼーション

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

神経科学

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.

ヒト従来型骨肉腫からのがん幹細胞培養法の確立

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.

がんの確立は、ヒト従来の骨肉腫からの幹細胞培養物

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

がん研究

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.

RNA In Situハイブリダイゼーションアッセイによる形態学的組織状況における遺伝的変異、ショートターゲット、および点変異の可視化

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.

遺伝的変異、短期目標と交配試験場でRNA の形態学的組織コンテキストに点突然変異を可視化

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

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.

マウス卵子のmRNAを定量および局在化するための単一分子蛍光In SituハイブリダイゼーションSM-FISHの使用

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.

マウス卵母細胞における定量化するため、Localize Mrna に単一分子蛍光そのままの交配 (SM 魚) の使用

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

遺伝学

RNA完全性を備えた脱灰FFPE骨組織サンプルを調製するためのプロトコル 

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.

著者スポットライト:空間トランスクリプトミクスによる骨肉腫の高度な治療標的の探索

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.

骨組織の空間的トランスクリプトミクスを可能にする方法

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

発生生物学

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.

RNAシーケンシングを用いた、In vitroがんモデルにおける薬剤耐性に関連する新規スプライス変異体の検出

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.

に薬剤耐性に関連する新規スプライス変異体を検出するために、RNA配列決定を使用して、インビトロがんモデル

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