单分子分辨率下树突状RNA的荧光原位成像
Fluorescence In Situ Imaging of Dendritic RNAs at Single-Molecule Resolution.
作者信息
Batish Mona, Tyagi Sanjay
机构信息
Department of Medical and Molecular Sciences and Department of Biological Sciences, University of Delaware, Newark, Delaware.
Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, New Jersey.
出版信息
Curr Protoc Neurosci. 2019 Sep;89(1):e79. doi: 10.1002/cpns.79.
Abstract
RNA localization is an important step in gene regulation. Imaging RNAs in fixed and live cells provides contextual information about RNA distribution in the cells. Here, we provide detailed protocols for performing single-molecule fluorescence in situ hybridization (smFISH). smFISH detects mRNA molecules at single-molecule resolution in fixed neuronal cells using ∼50 small oligonucleotide probes for each mRNA. The technique has been successfully applied to understand RNA localization and distribution in various biological systems, ranging from Drosophila to the mammalian nervous system. The probes are small enough to bind to structured RNAs or RNAs that are part of RNA-protein complexes, thereby accounting for ∼85% of the total RNA; this enables a level of sensitivity equivalent to that of quantitative real-time PCR, but with anatomical resolution. © 2019 by John Wiley & Sons, Inc.
摘要
RNA定位是基因调控中的重要一步。对固定细胞和活细胞中的RNA进行成像可提供有关RNA在细胞中分布的背景信息。在此，我们提供了用于进行单分子荧光原位杂交（smFISH）的详细方案。smFISH使用针对每个mRNA的约50个小寡核苷酸探针，以单分子分辨率检测固定神经元细胞中的mRNA分子。该技术已成功应用于理解从果蝇到哺乳动物神经系统等各种生物系统中的RNA定位和分布。这些探针足够小，可以与结构化RNA或作为RNA-蛋白质复合物一部分的RNA结合，从而占总RNA的约85%；这使得灵敏度水平与定量实时PCR相当，但具有解剖学分辨率。© 2019约翰威立国际出版公司。
相似文献
1
Fluorescence In Situ Imaging of Dendritic RNAs at Single-Molecule Resolution.单分子分辨率下树突状RNA的荧光原位成像
Curr Protoc Neurosci. 2019 Sep;89(1):e79. doi: 10.1002/cpns.79.
2
Design, Labeling, and Application of Probes for RNA smFISH.RNA smFISH 探针的设计、标记与应用。
Methods Mol Biol. 2022;2537:173-183. doi: 10.1007/978-1-0716-2521-7_10.
3
Single mRNA Molecule Detection in Drosophila.果蝇中单个信使核糖核酸分子的检测
Methods Mol Biol. 2018;1649:127-142. doi: 10.1007/978-1-4939-7213-5_8.
4
Locating RNAs in situ with FISH-STIC probes.使用FISH-STIC探针原位定位RNA。
Methods Mol Biol. 2015;1206:137-48. doi: 10.1007/978-1-4939-1369-5_12.
5
Single-molecule resolution fluorescent in situ hybridization (smFISH) in the yeast S. cerevisiae.酿酒酵母中的单分子分辨率荧光原位杂交（smFISH）。
Methods Mol Biol. 2013;1042:33-46. doi: 10.1007/978-1-62703-526-2_3.
6
RNA Imaging with Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH).基于多重抗误差荧光原位杂交（MERFISH）的RNA成像
Methods Enzymol. 2016;572:1-49. doi: 10.1016/bs.mie.2016.03.020. Epub 2016 Apr 27.
7
Single Molecule RNA FISH (smFISH) in Whole-Mount Mouse Embryonic Organs.全胚胎小鼠器官中的单分子RNA荧光原位杂交（smFISH）
Curr Protoc Cell Biol. 2019 Jun;83(1):e79. doi: 10.1002/cpcb.79. Epub 2018 Nov 5.
8
Improved Methods for Single-Molecule Fluorescence In Situ Hybridization and Immunofluorescence in Caenorhabditis elegans Embryos.改进的秀丽隐杆线虫胚胎中单分子荧光原位杂交和免疫荧光方法。
Curr Protoc. 2021 Nov;1(11):e299. doi: 10.1002/cpz1.299.
9
Visualization and Quantification of Subcellular RNA Localization Using Single-Molecule RNA Fluorescence In Situ Hybridization.使用单分子 RNA 荧光原位杂交技术可视化和定量细胞内 RNA 定位。
Methods Mol Biol. 2022;2404:247-266. doi: 10.1007/978-1-0716-1851-6_13.
10
Quantitative Super-Resolution Imaging of Small RNAs in Bacterial Cells.细菌细胞中小RNA的定量超分辨率成像
Methods Mol Biol. 2018;1737:199-212. doi: 10.1007/978-1-4939-7634-8_12.
引用本文的文献
1
Single-cell and spatial transcriptomics map senescent vascular cells in arterial remodeling during atherosclerosis in mice.单细胞和空间转录组学绘制了小鼠动脉粥样硬化过程中动脉重塑过程中的衰老血管细胞图谱。
Nat Aging. 2025 Jul 14. doi: 10.1038/s43587-025-00889-z.
2
Enhanced packaging of U6 small nuclear RNA and splicing-related proteins into extracellular vesicles during HIV infection.在HIV感染期间，U6小核RNA和剪接相关蛋白被增强包装到细胞外囊泡中。
Sci Adv. 2025 Mar 14;11(11):eadq6557. doi: 10.1126/sciadv.adq6557. Epub 2025 Mar 12.
3
Recent Advancements in Imaging Techniques for Individual Extracellular Vesicles.个体细胞外囊泡成像技术的最新进展
Molecules. 2024 Dec 10;29(24):5828. doi: 10.3390/molecules29245828.
4
Simultaneous Protein and RNA Analysis in Single Extracellular Vesicles, Including Viruses.单细胞外囊泡（包括病毒）中的蛋白和 RNA 同步分析。
ACS Nano. 2024 Oct 1;18(39):26568-26584. doi: 10.1021/acsnano.4c03679. Epub 2024 Sep 22.
5
A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma.lnc-HLX-2-7、HLX 和 MYC 之间治疗靶点阳性反馈环促进 3 组髓母细胞瘤。
Cell Rep. 2024 Mar 26;43(3):113938. doi: 10.1016/j.celrep.2024.113938. Epub 2024 Mar 8.
6
The Piezo channel is a mechano-sensitive complex component in the mammalian inner ear hair cell.Piezo 通道是哺乳动物内耳毛细胞中的机械敏感复杂组件。
Nat Commun. 2024 Jan 16;15(1):526. doi: 10.1038/s41467-023-44230-x.
7
Enhanced myogenesis through lncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter.通过 lncFAM 介导的 HNRNPL 募集到 MYBPC2 启动子增强肌生成。
Nucleic Acids Res. 2022 Dec 9;50(22):13026-13044. doi: 10.1093/nar/gkac1174.
8
CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs.环形RNA荧光原位杂交技术：一种同时对线状RNA和环形RNA进行成像的新方法。
Cancers (Basel). 2022 Jan 15;14(2):428. doi: 10.3390/cancers14020428.
9
Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems.环状寡聚腺苷酸与转录因子 Csa3 结合的结构基础概述了 III 型和 I 型 CRISPR 系统之间的串扰。
J Biol Chem. 2022 Feb;298(2):101591. doi: 10.1016/j.jbc.2022.101591. Epub 2022 Jan 14.
10
Identification of a New Transcriptional Co-Regulator of STEAP1 in Ewing's Sarcoma.鉴定尤文肉瘤中 STEAP1 的新转录共调节因子。
Cells. 2021 May 24;10(6):1300. doi: 10.3390/cells10061300.
本文引用的文献
1
ClampFISH detects individual nucleic acid molecules using click chemistry-based amplification.钳位荧光原位杂交技术（ClampFISH）利用基于点击化学的扩增方法来检测单个核酸分子。
Nat Biotechnol. 2018 Nov 12. doi: 10.1038/nbt.4286.
2
Spatial organization of the somatosensory cortex revealed by osmFISH.osmFISH 揭示的躯体感觉皮层的空间组织
Nat Methods. 2018 Nov;15(11):932-935. doi: 10.1038/s41592-018-0175-z. Epub 2018 Oct 30.
3
Analysis of the androgen receptor-regulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression.分析雄激素受体调控的长链非编码 RNA 图谱，鉴定出 ARLNC1 在前列腺癌进展中的作用。
Nat Genet. 2018 Jun;50(6):814-824. doi: 10.1038/s41588-018-0120-1. Epub 2018 May 28.
4
Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy.MERFISH 和扩展显微镜技术对高密度 RNA 文库进行多重成像。
Sci Rep. 2018 Mar 19;8(1):4847. doi: 10.1038/s41598-018-22297-7.
5
FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry.FISH-Flow，一种使用荧光原位杂交和流式细胞术同时检测单细胞中mRNA和蛋白质的方法。
Nat Protoc. 2017 Jun;12(6):1245-1260. doi: 10.1038/nprot.2017.039. Epub 2017 May 18.
6
The long noncoding RNA acts as an intranuclear organizing hub for pre-mRNA molecules.长非编码 RNA 充当前体 mRNA 分子的核内组织中心。
Sci Adv. 2017 May 3;3(5):e1602505. doi: 10.1126/sciadv.1602505. eCollection 2017 May.
7
Astrocyte-produced leukemia inhibitory factor expands the neural stem/progenitor pool following perinatal hypoxia-ischemia.星形胶质细胞产生的白血病抑制因子在围产期缺氧缺血后可扩大神经干细胞/祖细胞库。
J Neurosci Res. 2016 Dec;94(12):1531-1545. doi: 10.1002/jnr.23929. Epub 2016 Sep 23.
8
Profiling stem cell states in three-dimensional biomaterial niches using high content image informatics.利用高内涵图像信息学分析三维生物材料微环境中的干细胞状态。
Acta Biomater. 2016 Nov;45:98-109. doi: 10.1016/j.actbio.2016.08.052. Epub 2016 Aug 31.
9
Single-Molecule RNA In Situ Hybridization (smFISH) and Immunofluorescence (IF) in the Drosophila Egg Chamber.果蝇卵室中的单分子RNA原位杂交（smFISH）和免疫荧光（IF）
Methods Mol Biol. 2015;1328:125-36. doi: 10.1007/978-1-4939-2851-4_9.
10
Transcriptional expression of myelin basic protein in oligodendrocytes depends on functional syntaxin 4: a potential correlation with autocrine signaling.少突胶质细胞中髓鞘碱性蛋白的转录表达依赖于功能性 Syntaxin 4：与自分泌信号传导的潜在关联。
Mol Cell Biol. 2015 Feb;35(4):675-87. doi: 10.1128/MCB.01389-14. Epub 2014 Dec 15.
Zotero 插件