Name: rna fluorescence in situ hybridization for long non-coding rna localization in human osteosarcoma cells
Uploaded: 2023-06-16T14:30:00.000+00:00
Duration: 5 min 27 s
Description: Watch this Scientific Journal Video about RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells at JoVE.com

이 연구는 (1) 중국 국가 핵심 R&D 프로그램(2020YFE0201600)의 보조금으로 지원됩니다. (2) 국립자연과학재단(National Nature Science Foundation, 81973877 및 82174408) (3) 병원 TCM 준비의 산업 전환의 상하이 협력 혁신 센터; (4) Shanghai University of Traditional Chinese Medicine 예산 내 연구 프로젝트(2021LK047).

이 프로토콜에 사용되는 모든 재료, 시약 및 기기에 대한 자세한 내용은 재료 표를 참조하십시오. 그림 1 은 RNA FISH에 대한 전체 프로토콜을 보여줍니다. 표 1 에는 모든 용액의 조성이 포함되어 있고 표 2 에는 이 프로토콜에 사용된 프라이머 서열이 포함되어 있습니다.
1. 프로브 준비
<ol><li>예를 들어 GenBank(https://www.ncbi.nlm.nih.gov/genbank/)에서 관심 표적 lncRNA의 FASTA 염기서열을 식별하고 획득합니다. 웹 사이트의 표시에 따라 온라인으로 ISH 프로브를 설계하고19 설계 알고리즘의 제안을 검토하여 Cy3 라벨로 주문할 프로브를 나열합니다.</li>
	<li>혼성화 완충액을 37°C에서 2시간 전에 배양합니다.</li>
	<li>4 광학 밀도(OD) 프로브를 160μL의 디에틸피로카보네이트(DEPC) 처리된 ddH2O에 1mg/mL 농도로 빛에서 멀리 떨어뜨립니다. 참고: 광학 밀도(OD)는 DNA와 RNA의 측정 단위를 나타냅니다. 일반적으로 1 OD 단위 = 33 μg/mL DNA.</li>
	<li>각 웰에 대해 200μL의 프로브 혼합물(프로브 1.2μL-10μL, 혼성화 완충액 70μL, DEPC 처리된 ddH 2O 내지 200μL로 부피 구성)을 준비하고, 50μg/mL, 25μg/mL, 12.5μg/mL 및 6μg/mL 시리즈를 설정합니다. 알림: 프로브 농도는 사전에 실험적으로 탐색해야 합니다. 높은 프로브 농도는 lncRNA의 비특이적 결합으로 이어지고, 낮은 프로브 농도는 lncRNA의 둔감하거나 검출 실패로 이어집니다.</li>
	<li>프로브 혼합물을 73°C에서 5분 동안 변성시킵니다. 참고: Cy3 표지 DNA 프로브가 이중 가닥인 경우 프로브를 95°C에서 5분 동안 단일 가닥 DNA로 변성시킨 다음 얼음에서 2분 동안 빠르게 냉각합니다.</li>
</ol>2. 세포 준비
<ol><li>12웰 세포 배양 플레이트의 멸균 유리 커버슬립에 웰당 50,000개의 143B 세포를 파종하고 DMEM에서 24시간(37°C, 5% CO2) 동안 배양합니다. 참고: 여기에 파종된 특정 세포 수는 세포 크기에 따라 다르며, 이는 파종된 세포가 24시간 동안 배양된 후 50%의 합류점에 도달하는 데 적합합니다. 멸균 유리 커버슬립은 12웰 플레이트에 적합하도록 둥글게 되어 있습니다.</li>
	<li>배지를 제거하고 2x 인산염 완충 식염수(PBS)로 5 x 1분 동안 세척합니다. 참고: DEPC 처리된 ddH2O로 PBS 1개를 준비합니다. DEPC 처리가 없는 PBS는 RNase를 함유하고 있기 때문에 사용할 수 없습니다. RNase가 없는 조건에서 다음 단계를 모두 수행합니다.</li>
	<li>1x PBS를 제거하고 각 웰에 200μL의 100% 에탄올을 추가하여 실온에서 15분 동안 고정합니다.</li>
	<li>에탄올을 제거하고 각 웰에 200μL의 0.1% Triton X-100(1x PBS 기준)을 넣고 실온에서 15분 동안 배양합니다. 참고: 시간은 Triton X-100 투과성으로 엄격하게 제어해야 하며 너무 길어서는 안 됩니다.</li>
	<li>0.1% Triton X-100을 제거하고 1x PBS로 2 x 5분 동안 세척합니다. 참고: 여기서 프로토콜을 일시 중지해야 하는 경우 PBS를 70% 에탄올로 교체하고(100% 에탄올을 RNase가 없는 ddH2O로 희석) 샘플을 4°C에서 최대 3개월 동안 보관합니다.</li>
	<li>1x PBS를 제거하고, 200μL의 2x 식염수 나트륨(SSC) 완충액(RNase가 없는 ddH 2O로20xSSC 희석)을 각 웰에 넣고 37°C에서 30분 동안 배양합니다.</li>
	<li>2x SSC 버퍼를 제거하고 각 웰에 200μL의 70% 에탄올을 넣고 실온에서 3분 동안 배양합니다.</li>
	<li>70% 에탄올을 버리고 200μL의 85% 에탄올(RNase가 없는 ddH2O로 100% 에탄올 희석)을 각 웰에 넣고 실온에서 3분 동안 배양합니다.</li>
	<li>85% 에탄올을 버리고 각 웰에 200μL의 100% 에탄올을 넣고 실온에서 3분 동안 배양합니다.</li>
	<li>100% 에탄올을 흡수하고 버리십시오. 우물을 말리십시오.</li>
</ol>3. 형광 in situ hybridization (FISH)
<ol><li>200μL의 프로브 혼합물(1.5단계에서와 같이 변성됨)을 각 웰에 추가하고 37°C에서 밤새 배양합니다(16-18시간). 알림: 혼성화 온도와 프로브 농도 사이에는 강한 양의 상관 관계가 있습니다. 따라서 배경을 최적화하려면 혼성화 온도와 프로브 농도를 모두 줄여야 합니다.</li>
	<li>다음날 37 °C에서 샘플을 꺼내 프로브 혼합물을 폐기합니다. 각 웰에 0.4x SSC/0.3% Tween-20 완충액 200μL를 추가하고(65°C에서 예열) 실온에서 2분 동안 세척합니다.</li>
	<li>0.4x SSC/0.3% Tween-20 완충액을 제거하고 각 웰에 2x SSC/0.1% Tween-20 완충액 200μL를 추가한 다음 실온에서 2분 동안 세척합니다.</li>
	<li>2x SSC/0.1% 트윈-20 버퍼를 제거하고 200μL의 4',6-디아미디노-2-페닐린돌(DAPI) 염색 용액(1μg/mL)을 추가하고 빛을 피해 20분 동안 염색합니다.</li>
	<li>DAPI 염료 용액을 버리고 실온에서 1x PBS로 2분 동안 세척합니다.</li>
	<li>슬라이드에 껌이 들어 있는 장착 매체 50μL를 추가하고 유리 커버슬립을 슬라이드에 올려 고정합니다. 알림: 커버슬립은 셀 면이 아래로 향하게 하여 슬라이드에 놓아야 합니다.</li>
	<li>형광 현미경으로 관찰하십시오. 알림: 형광 현미경 또는 레이저 컨포칼 현미경을 사용하십시오. 후자는 더 높은 감도와 선명도 이미징을 생성합니다.</li>
</ol>

인간 골육종 세포에서 Long Non-coding RNA 국소화를 위한 FISH

<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>

저자는 경쟁하는 재정적 이익이 없다고 선언합니다.

이 RNA FISH 프로토콜은 세포에서 lncRNA의 국소화를 검출할 수 있을 뿐만 아니라 세포에서 다른 RNA, DNA 또는 단백질의 공동 국소화를 검출할 수 있으며, 이는 파라핀 포매 조직에서 lncRNA의 위치를 검출하는 데에도 사용할 수 있습니다. 그러나, 이러한 경우에서의 구체적인 프로토콜은 상이한데, 그 이유는 파라핀-포매 조직이 탈왁스될 필요가 있기 때문이다21. 이 실험 절차는 48웰 또...

인간 게놈에 대한 우리의 이해는 최근 전체 게놈 기술의 발전으로 크게 확장되었습니다. 인간 게놈의 약 93%는 RNA로 전사될 수 있지만 RNA의 2%만 단백질로 번역될 수 있습니다. 단백질 번역 기능이 없는 나머지 98%의 RNA를 non-coding RNA(ncRNA)라고 합니다1. 200개 이상의 뉴클레오티드를 포함하는 긴 ncRNA(noncoding RNA(ncRNA)의 한 종류인 긴 ncRNA(lncRNA)는 분화, 주기 조절, 세포자멸사, 이동 및 침습과 같은 세포의 많은 생리학적 및 병리학적 과정에 관여하기 때문에 점점 더 주목을 받고 있습니다 3,4,5. LncRNA는 염색질 구조 및 핵 유전자 발현 조절, mRNA 접합 과정 제어, 전사 후 변형 등 다양한 메커니즘을 통해 역할을 수행합니다6. LncRNA는 전사 및 전사 후 수준 모두에서 악성 종양의 발생, 발달 및 전이를 조절합니다. 전사 조절은 염색체 구조에 대한 결합을 통해 RNA 전사에 영향을 미침으로써 핵에서 실현되는 반면, 전사 후 조절은 내인성 경쟁 RNA(ceRNA) 메커니즘을 통해 표적 유전자를 제어하여 세포질에서 실현됩니다 5,7,8. CeRNA는 RNA 상호 작용의 새로운 메커니즘, 즉 lncRNA가 miRNA를 흡착하고 관련 표적 유전자의 miRNA 매개 분해를 억제하는 스폰지 역할을 할 수 있음을 밝혔습니다9. 그러므로, lncRNAs의 subcellular 국소화에 관하여 정보는, 특정한 lncRNA가 세포질 또는 핵에서 있다는 것을, 그들의 생물학 기능을 확인하는 것을 돕도록 중요합니다.
현재 lncRNA 국소화는 주로 두 가지 방법으로 검출되는데, 하나는 핵/세포질 분획 분리 분석에 의한 것이고 다른 하나는 RNA FISH에 의한 것입니다. 전자에서는 세포질 및 핵 분획의 RNA를 각각 추출한 다음 특정 lncRNA 프라이머로 PCR 증폭을 수행하여 세포질과 핵에서 lncRNA의 비율을 검출합니다. 이 방법의 장점은 시간 효율성인 반면, 단점은 실제 lncRNA 국소화가 세포질과 핵에서 lncRNA의 상대적 비율에 의해 직접 반영되지 않는다는 것입니다. RNA FISH는 형광 염료10으로 표지한 후 lncRNA 특이적 안티센스 사슬 서열의 설계를 통해 세포에서 lncRNA 국소화를 검출할 수 있습니다. RNA FISH 분석법은 형광단 표지된 다중 올리고 프로브 세트(11), LNA 프로브(12) 및 분지형 DNA(bDNA) 프로브(13)를 포함한 프로브 기술 및 검출 방법의 발전으로 개선되었습니다. RNA FISH는 lncRNA의 국소화를 검출할 수 있을 뿐만 아니라 이중색 또는 다색 면역형광을 사용하여 다른 RNA, DNA 또는 단백질의 공동 국소화도 검출할 수 있습니다14.
이 연구에서는 RNA FISH에 의한 골육종 세포(143B)에서 lncRNA small nucleolar RNA host gene 6(SNHG6)의 상세한 세포 내 국소화 검출 프로토콜을 예로 포함했습니다. SNHG6는 성숙한 접합 형태의 600-730 뉴클레오티드 lncRNA이며 대장암, 위암, 난소 투명 세포 암종, 골육종 및 간세포 암종을 포함한 다양한 인간 암에서 새로운 종양 유전자로 확인되었습니다15,16,17,18. 연구에 따르면 SNHG6는 증식, EMT 및 자가포식과 같은 암세포의 생물학적 행동에 관여하는 것으로 확인되었으며, miRNA15,16,17에 결합(스폰지)하여 표적 유전자에 영향을 미칠 수 있는 SNHG6의 세포질 국소화를 보여주었습니다. RNA FISH에 의한 SNHG6 세포내 국소화의 상세한 검출 프로토콜이 본원에 제시되어 있다.

<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

암세포의 증식, 상피-중간엽 전이(EMT), 이동, 침투 및 자가포식을 조절하는 것과 같은 암에서 긴 비암호화 RNA(lncRNA)의 중요한 역할이 연구되었습니다. 세포에서 lncRNA의 국소화 검출은 세포의 기능에 대한 통찰력을 제공할 수 있습니다. 형광 염료로 표지한 후 lncRNA 특이적 안티센스 사슬 서열을 설계함으로써 RNA 형광 in situ hybridization(FISH)을 적용하여 lncRNA의 세포 국소화를 검출할 수 있습니다. 현미경 검사법의 발달과 함께, RNA 물고기 기술은 지금 잘 표현되지 않는 lncRNAs의 구상 조차 허용합니다. 이 방법은 lncRNA의 국소화를 단독으로 검출할 수 있을 뿐만 아니라 이중색 또는 다색 면역형광을 사용하여 다른 RNA, DNA 또는 단백질의 공동 국소화도 검출할 수 있습니다. 여기에서는 인간 골육종 세포(143B)에서 lncRNA small nucleolar RNA host gene 6 (SNHG6)을 예로 들어 RNA FISH의 상세한 실험 조작 절차 및 주의사항을 포함하여, RNA FISH 실험, 특히 lncRNA FISH를 수행하고자 하는 연구자들에게 참고자료를 제공하였다.

인간 골육종 세포에서 SNHG6 FISH의 대표적인 이미지가 표시됩니다(그림 2). 네거티브 컨트롤은 네거티브 Ctrl 프로브로 처리됩니다. 양성 대조군은 U6 프로브 20으로 처리됩니다. SNHG6 프로브와 U6 프로브는 적색 형광을 방출하는 Cy3로 표지되어 있습니다. DAPI는 청색 형광을 방출하는 DNA를 염색하는 염료입니다. 이 결과는 SNHG6가 주로 세포질에 국한되어 있음을 ?...

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

저자 스포트라이트: 골육종 세포에서 lncRNA-SNHG6를 찾기 위한 RNA FISH 

본 프로토콜은 인간 골육종 세포에서 lncRNA를 국소화하기 위한 RNA 형광 in situ 교잡의 방법을 기술한다.

인간 골육종 세포에서 긴 비코딩 RNA 국소화를 위한 RNA Fluorescence In Situ Hybridization

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

우리 연구의 범위는 RNA FISH 실험을 수행하려는 과학자들에게 참조를 제공하는 상세한 RNA FISH 프로토콜을 제공하는 것입니다. 우리의 연구는 RNA FISH가 인간 골육종 세포에서 lncRNA를 국소화하는 데 사용될 수 있음을 확인했습니다. 당사의 프로토콜은 인간 골육종 세포에서 lncRNA-SNHG6를 참조로 사용하여 RNA FISH에 대한 포괄적인 실험 절차와 예방 조치를 제공했습니다.

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

Research

JoVE Journal

Cancer Research

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

1.3K 조회수.  Shanghai University of Traditional Chinese Medicine. 우리 연구의 범위는 RNA FISH 실험을 수행하려는 과학자들에게 참조를 제공하는 상세한 RNA FISH 프로토콜을 제공하는 것입니다. 우리의 연구는 RNA FISH가 인간 골육종 세포에서 lncRNA를 국소화하는 데 사용될 수 있음을 확인했습니다. 당사의 프로토콜은 인간 골육종 세포에서 lncRNA-SNHG6를 참조로 사용하여 RNA FISH에 대한 포괄적인 실험 절차와 예방 조치를 제공했습니다.

비디오: 저자 스포트라이트: 골육종 세포에서 lncRNA-SNHG6를 찾기 위한 RNA FISH 

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

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

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

암의 설립은 인간 기존의 골육종에서 세포 배양 줄기

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

연구

암 연구학

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.

Murine Oocytes에 계량 및 방화 mRNAs 단일 분자 형광에서 제자리 교 잡 (SM-물고기)의 사용

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

유전학

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.

전이율을 제어하는 절단을 가진 신제닉 직소 성 골육종 스프라그 Dawley 쥐 모델

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.

머릿속 RNA 현지화에서 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 ...

생물학

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.

고해상도를 사용하여 뇌 섹션에서 Axonally 지역화 된 mRNA를 검출<em&gt; 현장에서</em&gt; 하이브리드

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

신경과학

RNA 무결성을 갖춘 탈염화된 FFPE 뼈 조직 샘플을 준비하기 위한 프로토콜 

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.

저자 스포트라이트: Exploring Advanced Therapeutic targets in Osteosarcoma Through Spatial Transcriptomics(공간 전사체학을 통한 골육종의 고급 치료 표적 탐색)

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.

뼈 조직의 공간 전사체학(spatial transcriptomics)을 가능하게 하는 방법

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

발생학

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.

방사성 현장에서</em조직에서 다양한 유전자 발현 패턴을 검출> 하이브리드화

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.

인간 골육종 세포에서 긴 비코딩 RNA 국소화를 위한 RNA Fluorescence In Situ Hybridization

필기록

요약

더 많은 비디오 탐색

이 비디오의 챕터

암의 설립은 인간 기존의 골육종에서 세포 배양 줄기

Murine Oocytes에 계량 및 방화 mRNAs 단일 분자 형광에서 제자리 교 잡 (SM-물고기)의 사용

전이율을 제어하는 절단을 가진 신제닉 직소 성 골육종 스프라그 Dawley 쥐 모델

머릿속 RNA 현지화에서 Xenopus Oocytes

고해상도를 사용하여 뇌 섹션에서 Axonally 지역화 된 mRNA를 검출

뼈 조직의 공간 전사체학(spatial transcriptomics)을 가능하게 하는 방법

뼈 조직의 공간 전사체학(spatial transcriptomics)을 가능하게 하는 방법

뼈 조직의 공간 전사체학(spatial transcriptomics)을 가능하게 하는 방법

방사성 현장에서 하이브리드화

전체 마운트 RNA 형광