单分子荧光原位杂交实验中单个大肠埃希菌细胞的转录标记方法
Summary
这篇手稿描述了一种方法, 标记个别信使 RNA (mRNA) 转录与荧光标记的 DNA 探针, 用于分子荧光原位杂交 (smFISH) 实验在大肠杆菌. smFISH 是一种可视化方法, 它允许在固定的单个细胞中同时检测、定位和量化单个 mRNA 分子。
Abstract
一种方法被描述为标记单独信使 RNA (mRNA) 抄本在固定的细菌为使用在分子荧光原位杂交 (smFISH) 实验在大肠杆菌. smFISH 允许测量感兴趣基因的 mRNA 拷贝数的细胞变异, 以及转录的亚细胞位置。所涉及的主要步骤是固定的细菌细胞培养, 性的细胞膜, 和杂交的目标抄本与一套商业可用的短荧光标记寡核苷酸探针。smFISH 可以允许对同一细胞中的多个基因的转录进行成像, 并在不同荧光标记之间的频谱重叠施加限制。在下面所示的协议完成后, 细胞可以很容易地成像使用显微镜和适合低强度的荧光相机。这些图像, 连同从相衬框架的分割获得的细胞轮廓, 或从细胞膜染色, 允许计算的 mRNA 拷贝数分布的样本细胞使用开源或定制的软件。本文所描述的标记方法也可以应用于随机光学重建显微镜 (风暴) 的图像记录。
Introduction
随机性是基因表达的一个基本的和不可避免的方面, 并引起细胞细胞的异质性1, 无论是在转录和蛋白质水平上的2,3。在定义良好的条件下量化细胞间的变异性, 为基因表达及其调控的基本过程提供了一个独特的窗口。一个重要来源的细胞细胞异质性细菌发生在转录水平。成绩单的数量不仅因转录的随机性而异, 也因后的过程, 如由小 rna 和 RNAases2的调节。一种直接以定量方式访问这种异质性的方法是通过荧光标记特定基因在 smFISH 中的单个转录。这种方法允许检测和特定的 RNA 分子的亚细胞定位固定的, 个别的细菌单元4。基因与一组荧光标记的 ~ 20 基长的寡核苷酸杂交, 目的是选择性地绑定到感兴趣的记录5,6。多重标记可确保在背景荧光之上检测, 而单个 mRNA 分子在荧光显微镜下显示为衍射限制斑点7 (见图 1)。还有其他方法来标记 mRNA 分子, 其中互补齐聚物探针携带共轭半 (如, 生物素或辛), 被检测使用第二荧光标记的记者技术8。
除了 smFISH, 还有其他方法可以提供关于成绩单的定量信息。一些, 如北印迹或定量 PCR, 探针的体积, 从而可以测量既不是 mRNA 的数量, 也不是他们的位置在单个细胞。因此, 这些方法不适合量化细胞的变异性。最近的一种基于图像的技术, 允许定量的 rna 的副本数量和细胞内的位置, 称为复用误差鲁棒荧光原位杂交 (MERFISH) 已经发展。MERFISH 是基于一个独特的条形码的分配, 由固定数量的荧光标记的寡核苷酸探针的定义组合。这些条形码在 smFISH 测量的连续的圆读出来, 与漂白跟随每轮杂交, 从而增加吞吐量由二个数量级9,10。这种技术需要一个自动流体处理系统和适当的探头设置设计。
多重荧光标记的个人成绩单组合, 连同新的超分辨率技术, 如随机光学重建显微镜 (风暴)11, 使分辨率增加十倍转录的亚细胞定位。在风暴中, 一个合适的组合荧光探针和成像缓冲允许多个周期的荧光发射每个探针分子 (闪烁)。风暴也可用于图像的大肠杆菌转录和观察 RNA 的全基因组的空间组织, 通过标签同时所有的记录的兴趣,12。
上述所有的单细胞方法都是基于固定细胞的成像记录。因此, 他们没有提供任何关于细胞内转录物的动力学特性的信息。要跟踪活细胞的转录13, 基因可以标记的基因的融合的利益, 以一个数组的绑定网站。后者由 RNA 结合的蛋白质认可, 例如噬菌体 MS2 外套蛋白质, 被熔化对荧光蛋白质例如绿色萤光蛋白质 (GFP)10,14,15。
在这里, 我们描述一种方法, 标记个别基因与一组荧光标记的 DNA 探针, 用于 smFISH 实验, 特别是在大肠杆菌中使用。此外, 我们表明, 相同的标记方案可以用于风暴测量, 小修改。
Protocol
1. 探头设计 注: 本协议使用商业上可用的寡核苷酸探针已标记荧光。探针由一组与目标 mRNA 互补的特定序列组成, 每个探针被共轭到一个单一的荧光分子。或者, 可以将荧光标记附加到探测器上, 如其他地方所述5,16。 设计 smFISH 探针使用由阿金 Raj 开发的探针设计器16算法 (Oudenaarden 实验室, 马萨诸塞技术学?…
Representative Results
我们进行了 smFISH 测量的 galK 和 sodB 转录在大肠杆菌细胞。抄本与一组与目标序列互补的特定序列杂交, 每个探针被共轭到一个单一的荧光分子 (见材料表)。MG1655 野生型大肠杆菌菌株的荧光和相衬图像 (或 JW0740)19 galK-已删除的应变 (ΔgalK) 被暴露到2毫米 D-藻显示在图 1A和1B中。在接触 d-藻后, 细胞被固…
Discussion
我们在实验室测量了使用 smFISH 方法 2的大肠杆菌细胞中不同基因的转录数量。简而言之, 这个过程包括以下步骤: 细胞固定, 性的膜, 允许探针穿透, 探针杂交, 和样品成像使用标准的荧光显微镜。此过程基于以前发布的一些修改6,7,16。此前有报道称, smFISH 要求寡核苷酸探针的数量在48-72 范围内, 以达到在…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了以色列科学基金会赠款 514415 (对 BSF) 和一项非名 (NSF) 赠款2016707的支持。厄尔曼教授的支持, 也承认了。
Materials
| Dextran sulfate sodium salt | Sigma-Aldrich | D8906 | |
| Pure Ethanol, 99.5%, ACS reagent, absolute | Mallinckrodt Baker – Avantor | 8025.25 | |
| Diethylpyrocarbonate (DEPC) | Sigma-Aldrich | D5758 | |
| RNase-free 20X SSC | Life Technologies/Ambion | AM9763 | |
| RNase-free 10X PBS | Life Technologies/Ambion | AM9625 | |
| TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) BioUltra, for molecular biology | Sigma-Aldrich | 93283 | |
| nuclease-free water | Thermo Fisher Scientific | 10977035 | |
| Formaldehyde solution for molecular biology, 36.5-38% in water | Sigma-Aldrich | F8775 | |
| Deionized formamide, nuclease free | Thermo Fisher Scientific/Ambion | AM9342 | |
| E. coli tRNA (ribonucleic acid, transfer type xx from escherichia) | Sigma-Aldrich | R1753-500UN | |
| UltraPure BSA (50mg/ml) | Thermo Fisher Scientific/Ambion | AM2616 | |
| Vanadyl-ribonucleoside complex,VRC, 200 mM | New England Biolab | S1402S | |
| Poly-L-Lysine | Sigma | P4707 | |
| Cysteamine and oxygen | Sigma-Aldrich | 30070 | |
| Glucose Oxidase from Aspergillus niger, Type VII, 50KU | Sigma-Aldrich | G2133 | |
| Catalase | Sigma-Aldrich | C40 | |
| D-glucose | Sigma-Aldrich | G8270 | |
| D-fucose | Sigma-Aldrich | F8150 | |
| Vybrant DiO Cell-Labeling Solution | Life Technologies | V2286 | |
| Agarose,low melting reagent | Sigma-Aldrich | A9414 | |
| Adhesive silicone isolator 24-2mm Dia. X 1.8 mm depth JTR24R-A2-2.0 | Grace Bio-Labs | JTR24R-A2-2.0 666208 | |
| poly-D-lysine-coated glass bottom Glass Bottom Culture Dishes | MatTek Corporation | P35GC-1.5-14-C | |
| Super life nitrile powder free examination gloves | Supermax | TC-N-9889 | |
| Brand sterilization incubator tape | Sigma-Aldrich | BR61750 | |
| Microcentrifuge tubes (1.8 ml) | Axygen – Corning Life Sciences | MCT-175C | |
| Falcon round-bottom polypropylene tubes (14 ml) | BD Biosciences | 352059 | |
| Conical-bottom centrifuge polypropylene tubes (50 ml) | Corning | 430828 | |
| Serological pipettes (Corning 5 ml) | Corning Life Sciences | 4051 | |
| Serological pipettes (Corning 10 ml) | Corning Life Sciences | 4488 | |
| Serological pipettes (Corning 25 ml) | Corning Life Sciences | 4251 | |
| Spectrophotometer cuvettes | Sarstedt | 67.742 | |
| RNase-free pipette tips 0.2 – 20 μl | FroggaBio | FT20 | |
| RNase-free pipette tips 10 – 200 μl | Axigene/corning | TF-200 | |
| RNase-free pipette tips 100 – 1000 μl | FroggaBio | FT1000 | |
| RNase-free pipette tips 100 – 1000 μl | Sorenson | 14200 | |
| Syringe disposile 10 mL needle G-21 | Becton Dickinson, Biosciences | BD-309643 | |
| Minisart 0.2 um Syringe Filter | Sartorius | 16534 K | |
| Nikon instruments microscope type A immersion oil A, 8cc | Nikon | MXA20233 | |
| Microscope slides 76 x26, 3"x1"x1mm | Thermo Fisher Scientific | 421-004ET | |
| #0 coverslip slide 24×60 | Thermo Fisher Scientific/Menzel | BNBB024060A0 | |
| Orbital shaker | M.R.C | TOU-50 | |
| Hot block | M.R.C | ||
| Vortex | Fried Electric Company | G-560-E | |
| Microcentrifuge | Eppendorf | 5427R | |
| Centrifuge | Eppendorf | 5810R | |
| Portable Pipet-Aid XP2, Pipette Controller | Drummond Scientific Company | 4-000-501-I | |
| OD600 Spectrophotometer for Bacterial Growth Rates DiluPhotometer | Midsci | OD600-10 | |
| iXon X3 EMCCD camera | Andor | DU-897E-CS0-#BV | |
| Eclipse Ti microscope | Nikon | MEA53100 | |
| CFI plan apochromat DM 100X oil objective lambda PH-3 N.A 1.45 WD 0.13 | Nikon | MRD31905 | |
| Filter set (TRITC/CY3): EX – ET545/30X; EM – ET620/60M; BS – T570LP | Nikon | 49005 | |
| Filter set (CY5): EX – ET640/30X; EM – ET690/50M; BS – T660P | Nikon | 49009 | |
| Nis-elements AR auto reaserch software | Nikon | MQS31000 | |
| STORM microscope | Vutara | SR-200 | |
| NA 1.2 water immersion objective | Olympus | ||
| SRX image acquisition and analysis software | Vutara | ||
| Evolve 512 EMCCD camera | Photometrics | ||
| Stellaris® FISH Probes, Custom Assay with CAL Fluor® Red 590 Dye | Biosearch Technologies Inc | SMF-1083-5 | |
| Probe Sequence for galK mRNA: | |||
| gtgttttttctttcagactc | |||
| tagccaaatgcgttggcaaa | |||
| ctgaatggtgtgagtggcag | |||
| caccaatcaaattcacgcgg | |||
| acgaaaccgtcgttgtagtc | |||
| tgataatcaatcgcgcaggg | |||
| gtggtgcacaactgatcacg | |||
| acgcgaactttacggtcatc | |||
| gctgattttcataatcggct | |||
| gcatcgagggaaaactcgtc | |||
| gttttcatgtgcgacaatgg | |||
| cacgccacgaacgtagttag | |||
| ttacgcagttgcagatgttt | |||
| tccagtgaagcggaagaact | |||
| tgctgcaatacggttccgac | |||
| gtccagcggcagatgataaa | |||
| tgaccgttaagcgcgatttg | |||
| agcctacaaactggttttct | |||
| gcggaaattagctgatccat | |||
| aaggcatgatctttcttgcc | |||
| cagtgagcggcaatcgatca | |||
| tgggcatggaaactgctttg | |||
| gatgatgacgacagccacac | |||
| gggtacgtttgaagttactg | |||
| gtgttgtattcgctgccaac | |||
| ggtttcgcactgttcacgac | |||
| tggctgctggaagaaacgcg | |||
| ttcaatggtgacatcacgca | |||
| catgcgcaacagcgttgaac | |||
| ggcgttttcagtcagtatat | |||
| atacgtttcaggtcgccttg | |||
| tgagactccgccatcaactc | |||
| gaaatcatcgcgcatagagg | |||
| caatttgcggcacggtgatt | |||
| ttgacgatttctaccagagt | |||
| acctttgtcgccaatcacag | |||
| ggatcagcgcgacgatacag | |||
| atattgttcagcgacagctt | |||
| gtctctttaatacctgtttt | |||
| ctccttgtgatggtttacaa | |||
| Stellaris® FISH Probes, Custom Assay with Quaser Fluor® Red 670 Dye | Biosearch Technologies Inc | SMF-1083-5 | |
| Probe Sequence for sodB mRNA: | |||
| gtgttttttctttcagactc | |||
| tagccaaatgcgttggcaaa | |||
| ctgaatggtgtgagtggcag | |||
| caccaatcaaattcacgcgg | |||
| acgaaaccgtcgttgtagtc | |||
| tgataatcaatcgcgcaggg | |||
| gtggtgcacaactgatcacg | |||
| acgcgaactttacggtcatc | |||
| gctgattttcataatcggct | |||
| gcatcgagggaaaactcgtc | |||
| gttttcatgtgcgacaatgg | |||
| cacgccacgaacgtagttag | |||
| ttacgcagttgcagatgttt | |||
| tccagtgaagcggaagaact | |||
| tgctgcaatacggttccgac | |||
| gtccagcggcagatgataaa | |||
| tgaccgttaagcgcgatttg | |||
| agcctacaaactggttttct | |||
| gcggaaattagctgatccat | |||
| aaggcatgatctttcttgcc | |||
| cagtgagcggcaatcgatca | |||
| tgggcatggaaactgctttg | |||
| gatgatgacgacagccacac | |||
| gggtacgtttgaagttactg | |||
| gtgttgtattcgctgccaac | |||
| ggtttcgcactgttcacgac | |||
| tggctgctggaagaaacgcg | |||
| ttcaatggtgacatcacgca | |||
| catgcgcaacagcgttgaac | |||
| ggcgttttcagtcagtatat | |||
| atacgtttcaggtcgccttg | |||
| tgagactccgccatcaactc | |||
| gaaatcatcgcgcatagagg | |||
| caatttgcggcacggtgatt | |||
| ttgacgatttctaccagagt | |||
| acctttgtcgccaatcacag | |||
| ggatcagcgcgacgatacag | |||
| atattgttcagcgacagctt | |||
| gtctctttaatacctgtttt | |||
| ctccttgtgatggtttacaa |
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Cite This Article
Arbel-Goren, R., Shapira, Y., Stavans, J. Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments. J. Vis. Exp. (130), e56600, doi:10.3791/56600 (2017).