单分子鱼的转录活性等位基因单细胞分析
1GCC Center for Advanced Microscopy and Image Informatics, 2Department of Molecular and Cellular Biology,Baylor College of Medicine, 3Center for Translational Cancer Research, Institute of Biosciences and Technology,Texas A&M University, 4Department of Pharmacology and Chemical Biology,Baylor College of Medicine
Summary
单分子RNA荧光就地杂交（smFISH）是一种在单细胞水平上准确量化特定RNA水平和定位的方法。在这里，我们报告我们已验证的实验室协议，用于湿板凳处理、成像和图像分析，用于特定RNA的单细胞量化。
Abstract
基因转录是细胞生物学的一个基本过程，它允许细胞解释和响应内部和外部的线索。测量 mRNA 水平的传统大宗种群方法（北方污点、PCR 和 RNAseq）缺乏提供响应中细胞间变化信息的能力。通过单分子RNA荧光就地杂交（smFISH），可以实现精确的单细胞和过敏可视化和定量。RNA-FISH 通过将目标RNA与标记的寡核苷酸探头杂交来执行。这些数据可以以中/高吞吐量方式进行成像，并通过图像分析管道提供成熟和新生的RNA的定量数据，所有这些数据均在单个单元格级别上。多年来，实验室使用本处描述的模型系统对固定、渗透、杂交和成像步骤进行了优化，从而成功、稳健地对 smFISH 标签进行单细胞分析。样品制备和处理的主要目标是生成高质量的图像，其特点是信号与噪声比高，以减少误报并提供更准确的数据。在这里，我们提出一个协议，描述从样品制备到数据分析的管道，以及针对特定样本的建议和优化步骤。
Introduction
基因转录和翻译是生物学中心教条中涉及的两个主要过程，从DNA序列到RNA，再到蛋白质1。在这项研究中，我们专注于在转录过程中产生信使RNA（mRNA）。新生的RNA分子包括非编码内电和编码外子。在mRNA进入细胞质以在核糖体2、3上进行翻译之前，Introns被共同转录拼接出来。
传统上，mRNA 的测量是在细胞的批量群（数亿到数百万）中进行的，具有经典的测定方法，如 RT-qPCR 或北方印迹。虽然非常强大，但这些方法是有限的，因为它们不能提供对细胞间变异（表型异质性）、对转录活性等位基因数量的识别，以及也许更重要的是缺乏空间信息的见解。最近，允许单细胞RNA测序（RNAseq）的全基因组技术开始弥合成像方法和测序4之间的差距。但是，RNAseq 的检测效率相对较低，处理步骤导致空间信息完全丢失5。虽然单细胞RNASeq可以提供对同源细胞群中表型异质性的见解，但原位杂交（RNA-FISH）可以促进在空间水平上和在6、7等位基因基础上更全面地探索目标基因表达。
RNA FISH 是一种技术，用于检测和定位固定细胞中的目标RNA分子。与早期的艰苦方法不同，目前的最先进的RNA-FISH利用了与目标RNA序列互补的商用核酸探针，这些探针通过沃森-克里克碱基配对8杂交到目标上。早期的原位杂交技术涉及Gall和Pardue在1969年建立的类似协议，因为样品是用核酸探针处理的，核酸探针专门杂交到靶RNA9。最初，测定是使用放射性或色度检测进行的：然而，在20世纪80年代，发展荧光原位杂交（FISH）协议DNA鱼和后来的RNA鱼开辟了路径，走向更敏感的检测，并有可能复用10，11。
RNA FISH的进一步发展已导致能够检测和量化单RNA分子（smFISH）12，13。直接检测是探针本身被标记为氟磷的方法。smFISH 的一个挑战是，需要足够的杂交探头/目标，以促进荧光信号作为独特的衍射限制点的检测。为了解决这个问题，人们可以使用一组短的单链DNA寡核苷酸，补充到目标RNA8，12，14的不同区域。多个探针的结合会增加局部荧光密度，使RNA通过荧光显微镜可见为一个独特的高强度点。这种方法是有利的，因为探针组池中寡核苷酸的脱靶结合可以通过定量分析点大小和强度来区分真实信号和虚假的寡核苷酸结合，从而通过减少误报12来促进更准确的分析。
检测 mRNA 的替代方法是经典的 BAC 克隆基刻痕翻译方法和最近开发的分支 DNA 系统。在BAC克隆的刻痕转换方法中，要标记的DNA是酶刻痕，在暴露的3’端添加了一种新的核苷酸。DNA聚合酶的活性我添加了一个标记的核苷酸，导致所需的探针15，16。分支DNA技术涉及寡核苷酸探针，成对杂交到RNA目标，这些探针利用多个信号放大分子被放大。这种方法可以显著提高信号与噪声比，但放大可以扭曲精确的量化，因为荧光点在大小和强度上可能大相径庭。
作为此协议的模型系统，作为 NIEHS 超级基金研究计划的一部分，我们充分利用了该协议，以量化内分泌干扰化学物质在加尔维斯顿湾/休斯顿船舶通道的影响，我们使用雌激素受体 （ER） 阳性乳腺癌细胞系 MCF-7 与 ER 激动剂 17+ -estradiol （E2）7，18，19通宵治疗。E2调节许多ER靶点基因，包括原型ER靶基因GREB17，20，21，22。此处描述的 smFISH 协议使用一组两组针对 GREB1 的光谱分离探针集;一个杂交到外星，另一个到内电，允许测量成熟和新生的RNA7。然后，我们使用表观显微镜，结合去革命图像 smFISH 标记的样品，然后应用图像分析程序来量化每个细胞的活性等位基因和成熟RNA 的数量。
Protocol
为了确保最佳效果，本协议的湿实验室部分要求所有步骤都提供标准的无 RNase 预防措施。例如，非常鼓励使用过滤的移液器尖端、无菌容器和无 RNase 缓冲器。还建议使用 RNase 抑制剂，如瓦纳迪尔核糖核苷复合物，特别是对于低丰度目标RNA。 1. 细胞培养和实验设置 保持粘附的MCF-7乳腺癌细胞（ATCC #HTB-22）在T75组织培养瓶与苯酚红色免费（只需要激素刺激?…
Representative Results
为了通过 smFISH 分析荷尔蒙反应，例如，我们选择了用于高吞吐量检测的雌激素受体 （ER） 模型，以确定在参与 NIEHS 超级基金研究计划7的背景下，在环境灾难中存在内分泌干扰化学物质。在本实验中，粘附的MCF-7乳腺癌细胞用ER激动剂17+-雌二（E2，10 nM）或车辆（DMSO）治疗24小时。与GREB1电子（图1中的红色，图1中的红色）和外波（Quasar 570，图<strong class="xfi…
Discussion
所描述的 smFISH 方法基于先前发布的协议 7、12、14。在此协议中，我们解释了从湿实验室（包括播种密度、固定时间、渗透度和探针浓度）到成像和图像分析（包括种子密度、渗透度和探针浓度）优化的关键步骤，为有兴趣对基因转录进行单细胞分析的实验室提供了完整的实验管道。
为了促进单细胞分析?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
MAM、FS 和米高梅由 NIEHS（项目 4、P42ES027704、PI、Rusyn）资助。MAM、FS、RMM、米高梅和PKS由CPRIT资助的GCC高级显微镜和图像信息学中心（RP170719）提供支持。贝勒医学院的综合显微镜核心也提供成像支持，资金来自NIH（DK56338和CA125123）、CPRIT（RP150578）、丹·邓肯综合癌症中心和约翰·邓恩湾海岸化学基因组学联合会。
Materials
| 17β Estradiol | Sigma-Aldrich | E2257 | CAS Number 50-28-2 |
| Ambion SSC (20X) Buffer, Rnase-free | ThermoFisher Scientific | AM9770 | |
| Corning DMEM with L-Glutamine and 4.5 g/L Glucose | Fisher Scientific | MT10017CV | Manufactured by Invitrogen |
| DAPI | Sigma-Aldrich | D8417 | Without sodium pyruvate |
| Dextran Sulfate, 50% Solution Sterile | Sigma-Aldrich | 3730-100ML | |
| Dulbecco's Phosphate Buffered Saline Ca++/Mg++ | Corning | 21030CV | Manufactured by Calbiochem |
| Ethanol, 200 Proof (100%) | Fisher Scientific | 07-678-005 | |
| Falcon 24-Well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate | Corning | 353047 | Manufactured by Decon Laboratories |
| Fetal Bovine Serum (FBS), 500 mL | Fisher Scientific | 50-753-2978 | Manufactured by Gemini Bio Products |
| GE Healthcare DeltaVision LIVE High Resolution Deconvolution Microscope | GE Healthcare | ||
| Hyclone Water, Molecular Biology Grade | Fisher Scientific | SH3053802 | |
| Immersion Oil 1.516 | GE Healthcare Life Sciences | 29162940 | Manufactured by GE Healthcare Bio-Science |
| L-glutamine Solution | Fisher Scientific | MT25005Cl | Manufactured by Corning |
| MCF-7 cells | ATCC | HTB-22 | |
| Molecular Grade Formamide | Promega | PAH5051 | |
| Olympus 60x/1.42 NA Objective | Olympus | ||
| Parafilm | Bemis Company, Inc. | 52858-076 | Distributed by VWR |
| Paraformaldehyde, 16% Solution, EM Grade | Electron Microscopy Sciences | 15710 | |
| Penicilin-Streptomycin Solution | Fisher Scientific | MT3001Cl | Manufactured by Corning |
| Ribonucleoside Vanadyl Complex | VWR | 101229-716 | Manufactured by New England Biosciences |
| RNase Away | Ambion | 9780 | |
| Sodium pyruvate, 100 mM | Fisher Scientific | 11-360-070 | Manufactured by Gibco |
| Thermo Scientific Glass Coverslips, #1.5 | Fisher Scientific | 12-545-81P | |
| Triton X-100, 100 mL | Fisher Scientific | BP151-100 | Manufactured by Fisher BioReagents |
| Trypsin-EDTA Solution 0.25% | Sigma-Aldrich | T4049-500ML | |
| Vectashield Antifade Mounting Medium 10 mL | Fisher Scientific | NC9265087 | Manufactured by Vector Laboratories |
Referencias
- Russell, P. J. Gene Expression: Transcription. iGenetics: A Mendelian Approach. , 331-352 (2006).
- Darnell, J. E. Variety in the level of gene control in eukaryotic cells. Nature. 297, 365-371 (1982).
- Köhler, A., Hurt, E. Exporting RNA from the nucleus to the cytoplasm. Nature Reviews Molecular Cell Biology. 8, 761-773 (2007).
- Kolodziejczyk, A. A., Kim, J. K., Svensson, V., Marioni, J. C., Teichmann, S. A. The Technology and Biology of Single-Cell RNA Sequencing. Molecular Cell. 58, 610-620 (2015).
- Shah, S., Lubeck, E., Zhou, W., Cai, L. In situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus. Neuron. 92, 342-357 (2016).
- Halpern, K. B., et al. Bursty Gene Expression in the Intact Mammalian Liver. Molecular Cell. 58, 147-156 (2015).
- Stossi, F., et al. Estrogen-induced transcription at individual alleles is independent of receptor level and active conformation but can be modulated by coactivators activity. Nucleic Acids Research. 48, 1800-1810 (2020).
- Coassin, S. R., Orjalo, A. V., Semaan, S. J., Johansson, H. E. Simultaneous Detection of Nuclear and Cytoplasmic RNA Variants Utilizing Stellaris RNA Fluorescence In situ Hybridization in Adherent Cells. Methods in Molecular Biology. 1211, 189-199 (2014).
- Gall, J. G., Pardue, M. L. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proceedings of the National Academy of the Sciences of the United States of America. 63, 378-383 (1969).
- Bauman, J. G., Wiegant, J., Borst, P., van Duijn, P. A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome labelled RNA. Experimental Cell Research. 128, 485-490 (1980).
- Singer, R. H., Ward, D. C. Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with biotinated nucleotide analog. Proceedings of the National Academy of the Sciences of the United States of America. 79, 7331-7335 (1982).
- Femino, A. M., Fay, F. S., Fogarty, K., Singer, R. H. Visualization of single RNA transcripts in situ. Science. 280, 585-590 (1998).
- Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods. 5, 877-879 (2008).
- Orjalo, A. V., Johansson, H. E. Stellaris RNA Fluorescence In situ Hybridization for the Simultaneous Detection of Immature and Mature Long Noncoding RNAs in Adherent Cells. Methods in Molecular Biology. 1402, 119-134 (2016).
- Johnson, C. V., Singer, R. H., Lawrence, J. B. Chapter 3 Fluorescent Detection of Nuclear RNA and DNA: Implications for Genome Organization. Methods in Cell Biology. 35, 73-99 (1991).
- Levsky, J. M., Singer, R. H. Fluorescence in situ hybridization: Past, present and future. Journal of Cell Science. 116, 2833-2838 (2003).
- Wang, F., et al. RNAscope: A novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. Journal of Molecular Diagnostics. 14, 22-29 (2012).
- Stossi, F., et al. Defining estrogenic mechanisms of bisphenol A analogs through high throughput microscopy-based contextual assays. Chemistry and Biology. 21, 743-753 (2014).
- Szafran, A. T., Stossi, F., Mancini, M. G., Walker, C. L., Mancini, M. A. Characterizing properties of non-estrogenic substituted bisphenol analogs using high throughput microscopy and image analysis. PLoS One. 12, 1-19 (2017).
- Rae, J. M., et al. GREB1 is a critical regulator of hormone dependent breast cancer growth. Breast Cancer Research and Treatment. 92, 141-149 (2005).
- Heldring, N., et al. Estrogen receptors: How do they signal and what are their targets. Physiological Reviews. 87, 905-931 (2007).
- Hah, N., et al. A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells. Cell. 145, 622-634 (2011).
- Kadam, P., Bhalerao, S. Sample size calculation. International Journal of Ayurveda Research. 1, 55-57 (2010).
- Kocanova, S., et al. Activation of estrogen-responsive genes does not require their nuclear co-localization. PLoS Genetics. 6, (2010).
- Shaffer, S. M., Wu, M. T., Levesque, M. J., Raj, A. Turbo FISH: A Method for Rapid Single Molecule RNA FISH. PLoS One. 8, (2013).
- Kim, S. O., Kim, J., Okajima, T., Cho, N. J. Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy. Nano Convergence. 4, (2017).
- Shieh, T. M., et al. Application of ribonucleoside vanadyl complex (RVC) for developing a multifunctional tissue preservative solution. PLoS One. 13, 1-14 (2018).
- Waters, J. C. Accuracy and precision in quantitative fluorescence microscopy. Journal of Cell Biology. 185, 1135-1148 (2009).
- Vinegoni, C., Feruglio, P. F., Weissleder, R. High dynamic range fluorescence imaging. IEEE Journal of Selected Topics in Quantum Electronics. 25, (2019).
- Tam, R., Shopland, L. S., Johnson, C. V., McNeil, J. A., Lawrence, J. B. Application of RNA FISH for visualizing gene expression and nuclear architecture. FISH: A Practical Approach. , 93-117 (2002).
- Jonkman, J., Brown, C. M., Wright, G. D., Anderson, K. I., North, A. J. Tutorial: guidance for quantitative confocal microscopy. Nature Protocols. , (2020).
- Trcek, T., et al. Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nature Protocols. 7, 408-419 (2012).
- Kwon, S. Single-molecule fluorescence in situ hybridization: Quantitative imaging of single RNA molecules. BMB Reports. 46, 65-72 (2013).
- Sibarita, J. B. Deconvolution microscopy. Advcances in Biochemical Engineering/Biotechnology. 95, 201-243 (2005).
- Takei, Y., Shah, S., Harvey, S., Qi, L. S., Cai, L. Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH. Biophysical Journal. 112, 1773-1776 (2017).
- Moffitt, J. R., Zhuang, X. RNA Imaging with Multiplexed Error-Robust Fluorescence in situ Hybridization (MERFISH). Methods in Enzymology. 572, 1-49 (2016).
- Shah, S., et al. Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH. Cell. 174, 363-376 (2018).
- Xia, C., Babcock, H. P., Moffitt, J. R., Zhuang, X. Multiplexed detection of RNA using MERFISH and branched DNA amplification. Scientific Reports. 9, 1-13 (2019).
- Lubeck, E., Coskun, A. F., Zhiyentayev, T., Ahmad, M., Cai, L. Single-cell in situ RNA profiling by sequential hybridization. Nature Methods. 11, 360-361 (2014).
- Wang, G., Moffitt, J. R., Zhuang, X. Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy. Scientific Reports. 8, (2018).
- Wassie, A. T., Zhao, Y., Boyden, E. S. Expansion microscopy: principles and uses in biological research. Nature Methods. 16, 33-41 (2019).
- Zimmerman, S. G., Peters, N. C., Altaras, A. E., Berg, C. A. Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries. Nature Protocols. 8, 2158-2179 (2013).
- Kochan, J., Wawro, M., Kasza, A. Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH. Biotechniques. 59, 209-221 (2015).
Citar este artículo
Mistry, R. M., Singh, P. K., Mancini, M. G., Stossi, F., Mancini, M. A. Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH. J. Vis. Exp. (163), e61680, doi:10.3791/61680 (2020).