搜索

Single Molecule RNA FISH in Arabidopsis Root Cells
拟南芥根细胞中的单个RNA分子FISH   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al., 2016). This single molecule RNA fluorescence in situ hybridization (smFISH) uses multiple single-labelled oligonucleotide probes to bind target RNAs and generate diffraction-limited signals that can be detected using a wide-field fluorescence microscope. We adapted a recent version of this method that uses 48 fluorescently labeled DNA oligonucleotides (20 mers) to hybridize to different portions of each transcript (Raj et al., 2008). This approach is simple to implement and has the advantage that it can be readily applied to any genetic background.

Keywords: Single RNA molecules(单个RNA分子), Fluorescent in situ hybridization(荧光原位杂交), Gene expression(基因表达), Arabidopsis(拟南芥), Transcription(转录)

Background

While single molecule FISH has been developed to quantitatively measure mRNAs at the single cell level for cultured cells, tissue sections and whole-mount invertebrate organisms, this method was not optimized for use in single cells in plants. Fluorescence imaging in plants is considerably challenging due to endogenous autofluorescence of plant tissues. Here, we report a method to detect single RNA molecules in plants. We describe the detection and automated counting of single transcripts within cells of fixed Arabidopsis root squashes. This method generates isolated cells and single-cell layers, which together with the use of red and far-red dyes maximizes signal-to-noise ratio limiting background noise.

Materials and Reagents

  1. 1.5 ml microcentrifuge tube
  2. Sterile SterilinTM 10 cm square Petri dishes for plant growth media (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 109 )
  3. 22 x 22 mm No. 1 glass coverslips (VWR, catalog number: 631-0124 )
  4. Poly-L-Lysine slides (Sigma-Aldrich, catalog number: P0425 )
  5. Razor blades (Agar Scientific, catalog number: AGT586 )
  6. Parafilm M® sealing film (Bemis, catalog number: PM992 )
  7. Hybridization chamber
    Note: Although these are available commercially (Corning, catalog number: 2551 ) we used 10 cm square Petri dishes covered externally with a layer of black insulation tape (RS Components, catalog number: 494-405 ). A double layer of tissue (KCWW, Kimberly-Clark, catalog number: 7557 ) was then placed in the base and saturated with sterile water before slides were placed on top of a single layer of Parafilm (see Figure 1).


    Figure 1. Illustration of the hybridization chambers used for smFISH experiments

  8. Low stender-form preparation dishes (VWR, catalog number: 470144-866 ; or similar rimmed glass dish)
  9. Arabidopsis thaliana roots
  10. Sodium hypochlorite (NaClO) (VWR, BDH®, catalog number: CABDH7038-4L )
  11. Sucrose (Sigma-Aldrich, catalog number: S9378-1KG )
  12. Phytagel (Sigma-Aldrich, catalog number: P8169 )
  13. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  14. Nuclease-free phosphate buffered saline solution (PBS, 10x) pH 7.4 (Thermo Fisher Scientific, AmbionTM, catalog number: AM9624 )
  15. Liquid nitrogen
  16. Ethanol suitable for molecular biology
  17. Nuclease-free 20x saline-sodium citrate (20x SSC) (Thermo Fisher Scientific, AmbionTM, catalog number: AM9763 )
  18. Clear nail varnish (Electron Microscopy Sciences, catalog number: 72180 ; or similar)
  19. Dextran sulphate (Sigma-Aldrich, catalog number: RES2029D )
  20. Custom RNA FISH Stellaris® probe sets
    We used the online Stellaris Probe Designer to design our smFISH probe sets: https://www.biosearchtech.com/support/education/stellaris-rna-fish. We used a default masking level 2 to avoid general problematic RNA sequences and selected the maximum number of 48 probes, an oligo length of 20 nt and 2 nt minimum spacing between probes.
    We also performed TAIR BLAST searches (https://www.arabidopsis.org/Blast/index.jsp) for each probe sequence and considered the results collectively to ensure target specificity. We found Quasar 570 and Quasar 670 dyes equally suitable for imaging RNA in Arabidopsis root cells, however we were unable to detect RNA labelled with Fluorescein modified probes.
  21. Murashige & Skoog basal medium with vitamins (any equivalent source would be suitable) (PhytoTechnology Laboratories®, catalog number: M519 )
  22. Nuclease-free Tris-EDTA buffer solution (10 mM Tris-HCl, 1 mM EDTA pH 8) (Sigma-Aldrich, catalog number: 93283 )
  23. DAPI (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 )
  24. Glucose (Sigma-Aldrich, catalog number: 158968-500G )
  25. Tris HCl buffer 1 M pH 8, nuclease-free (Thermo Fisher Scientific, AmbionTM, catalog number: AM9855G )
  26. Glucose oxidase (Sigma-Aldrich, catalog number: G0543 )
  27. Bovine liver catalase (Sigma-Aldrich, catalog number: C3155 )
  28. Deionized formamide (Sigma-Aldrich, catalog number: F9037 )
  29. 1 N HCl (Sigma-Aldrich, catalog number: 71763 )
  30. 10% (v/v) bleach (Vortex 5-10% sodium hypochlorite, Procter & Gamble, UK) diluted in dH2O
  31. TE buffer (10 nM Tris-HCl, 1 mM EDTA, pH 8.0)
  32. Nuclease-free water - not DEPC treated (QIAGEN, catalog number: 129117 )
  33. Murashige and Skoog medium (see Recipes)
  34. Wash buffer (see Recipes)
  35. Hybridization solution (see Recipes)
  36. DAPI solution (see Recipes)
  37. Anti-fade GLOX buffer (minus enzymes) (see Recipes)
  38. Anti-fade GLOX buffer (containing enzymes) (see Recipes)

Equipment

  1. Forceps (Fisher Scientific, S MurrayTM, catalog number: E017/01 ; or similar)
  2. Coplin jar (Sigma-Aldrich, catalog number: S6016 ; or similar)
  3. Plant growth chamber (Panasonic Healthcare, Sanyo, catalog number: MLR-352H-PE ; or similar)
  4. Fume cupboard (Labconco, catalog number: 2247300 ; or similar)
  5. Laminar flow cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraguardTM ECO Clean Bench Floor Stands , catalog number: 50109313; or similar)
  6. Laboratory oven (Cole-Palmer, catalog number: WZ-52412-83 ; or similar)
  7. Orbital shaker (Cole-Palmer, catalog number: WZ-51700-13 ; or similar)
  8. Wide-field fluorescence microscope
    Note: We used an Andor iXon EM-CCD camera (Andor, model: iXon EMCCD Camera ) fitted to a Zeiss, Elyra PS-1 (Zeiss, model: Elyra PS.1 ), however similar images have also been obtained using a standard CCD camera optimized for low light level imaging.
  9. A high numerical aperture (> 1.3) and 60 or 100x oil-immersion objective
  10. Strong light source, such as a mercury or metal-halide lamp (Xenon or LEDs are typically not bright enough)
  11. Filter sets appropriate for the fluorophores

Software

  1. ImageJ software: http://rsbweb.nih.gov/ij/index.html (Schindelin et al., 2015)
  2. FISHcount: https://github.com/JIC-CSB/FISHcount
  3. Graphpad Prism Software or MS Excel

Procedure

  1. Plant growth and microscope slide preparation
    1. Surface sterilize seeds in 5% (v/v) sodium hypochlorite for 5 min in a 1.5 ml microcentrifuge tube (inverting ~10 times) and then rinse three times in sterile distilled water.
    2. Plate the seeds on to Murashige and Skoog medium (pH 5.8) supplemented with 1% (w/v) sucrose and 0.5% (w/v) phytagel.
    3. Stratify the seeds by incubating for 2 days at 4 °C in darkness. Then, seedlings were grown at 22 ± 1 °C, in vertically oriented Petri dishes for at least 5 days (16 h light and 8 h dark).

  2. Fixation and preparation of Arabidopsis roots (see Figure 2 for an overview)


    Figure 2. Protocol steps for smFISH in Arabidopsis root cells. See main text for details.
     
    1. Cut root tips from Arabidopsis seedlings (approx. 1 cm) whilst still on plates and place into a small glass dish containing freshly prepared 4% paraformaldehyde and incubate for 30 min at room temperature under a fume hood.
    2. Remove the roots from the fixative and wash twice with 1x PBS.
    3. Arrange 3-4 roots onto a slide and cover with a coverslip. Gently squash each root onto the slide using either your thumb and be careful to avoid breaking the coverslip. Aim to splay the roots sufficiently to produce multiple files and isolated cells in a single cell layer (see Figure 3).


      Figure 3. Low magnification image a root meristem squash. Nuclei stained with DAPI (blue). This method yields many single-layer cells that are either isolated or within files. Scale bar = 200 μm.

    4. Use tweezers to hold the squashed roots under the coverslip and immerse each slide in liquid nitrogen for ~5 sec. After removal from the nitrogen, ease a razor blade between the coverslip and the slide and flick the coverslip off.
    5. Leave samples to air dry at room temperature for a minimum of 30 min.
      Note: To avoid increased levels of autofluorescence do not leave to dry for longer than 2 h.
    6. Permeabilize the samples by immersing the slides into a Coplin jar containing 70% ethanol for a minimum of one hour at room temperature. Fixed roots can be stored at 2 to 8 °C in 70% ethanol up to a week prior to hybridization.

  3. Probe hybridization
    1. Remove slides from the ethanol and allow residual ethanol to evaporate at room temperature.
    2. Wash 2 times on the slide with wash buffer for 5 min.
    3. Prepare a probe solution by adding 1 μl of each required probe stock solution to 100 μl of hybridization solution (250 nM final concentration).
    4. Add 100 μl probe solution to each slide, cover with a coverslip to prevent evaporation and incubate in a humid chamber at 37 °C for a minimum of 4 h (or overnight) in the dark.

  4. Sample mounting
    1. Remove the cover slip and wash the samples 2 times in 200 μl wash buffer for 5 min on the slide.
    2. Immerse the slide in a Coplin jar containing ~30 ml of wash buffer and incubate for 30 min at 37 °C, in the dark.
    3. Remove the slide from the jar and add 100 μl of DAPI solution to the slide then incubate at 37 °C for 30 min in the dark.
    4. Aspirate the DAPI solution carefully with a pipette and rinse with ~100 μl 2x SSC.
    5. Remove 2x SSC and add 100 μl anti-fade GLOX buffer (minus enzymes). Leave it to equilibrate for 1-2 min.
    6. Remove anti-fade GLOX buffer (minus enzymes) and add 100 μl of anti-fade GLOX buffer (containing enzymes) to each slide.
    7. Cover the samples with a coverslip, remove excess anti-fade GLOX buffer (plus enzymes) and seal with nail varnish.
    8. Image the slides the same day to avoid sample drying and fluorophore fading.

  5. Imaging
    1. Images are acquired on a wide-field epifluorescence microscope, with an optical sectioning size of 0.2 μm per z-plane and spanning the entire volume of the cell (around 5 μm, depending on cell size). Exposure times will vary depending on your microscope setup (light source, objective, etc.). We acquired our images using a Zeiss Elyra PS with a 100x oil-immersion objective (1.46 NA) and a cooled electron multiplying-CCD (charge-coupled device) Andor iXon 897 camera (512 x 512, QE > 90%). For probes labeled with Quasar 570 an excitation line of 561 nm was used and signals were detected at 570-640 nm; for probes labelled with Quasar 670 an excitation line of 642 nm and signals were detected at 655-710 nm; for DAPI an excitation line of 405 nm and signals were detected at wavelengths of 420-480 nm. Typically, we used 30 msec exposure time for DAPI, and 200-300 msec for Quasar dyes.
    2. After image acquisition, Z-stacks can be reduced to a 2D dataset using the maximum projection command in ImageJ (Image/Stack/Z Project). For a detailed description of this step please refer to ImageJ User Guide available at: https://imagej.nih.gov/ij/docs/guide/user-guide.pdf. The maximum projection command will result in an image with diffraction-limited spots corresponding to single RNAs. When exonic probes are used in conjunction with intronic probes for the same gene, some spots will co-localize within nuclei, corresponding to nascent RNA molecules that were being transcribed at the time of fixation (Figure 4).


      Figure 4. Detection of FLC transcripts in single cells. A. Schematic of the probes used to detect FLC transcripts: intronic (red) and exonic (green). FLC exons are represented as thick lines and introns shown as thin lines. B. Representative image showing FLC mRNA (green) and FLC nascent RNA (red) expression in Arabidopsis thaliana root cells. The diffraction-limited spots in the cytoplasm correspond to single FLC mRNA molecules (green), and the intense spots co-localizing with intronic signals (red) correspond to transcription sites. Depending on the cell cycle stage one can detect up to 2 dots (G1 phase) or 4 dots (G2 phase) for the intronic signals. The nucleus is stained with DAPI and shown in blue. Scale bar = 5 μm.

Data analysis

  1. For our image analysis we used a publically available mRNA counting programme that had been optiimised for our experimental setup. Details about how to install this programme on a Mac computer can be found at https://github.com/JIC-CSB/FISHcount.
    Note: We recommend FISH-quant for researchers wishing to quantify FISH data on Windows machines (Mueller et al., 2013).
  2. Figure 6 shows examples of ‘RNA identification’, ‘Cell Segmentation’ and ‘mRNA Per Cell’ output images that are generated by FISHcount. After performing analysis to a whole dataset, we recommend manually inspecting each ‘mRNA Per Cell’ output image (Figure 6D) to ensure that the image analysis workflow has not generated inaccurate results through incorrect segmentation. A detailed explanation of the image analysis workflow can also be found the Methods section in Duncan et al., 2016 (Figure 5).
  3. Data can then recorded and plotted as shown in Figure 7 using GraphPad Prism or MS Excel.


    Figure 5. Image analysis workflow (adapted from Duncan et al., 2016)


    Figure 6. Automated image analysis of FLC mRNA. A. Representative maximum projection image of cells labeled with FLC mRNA probes (green). DNA labeled with DAPI (blue). B-D. Screen shots showing sequential detection steps used to determine positive mRNA signals. B. FLC mRNA spot locations; C. Cell Segmentation; D. Output image with number of mRNAs per cell. Scale bars = 5 μm.


    Figure 7. Frequency distribution of FLC mRNA molecules per cell. A total of 520 cells were analyzed from three experiments. Error bars are ± SEM.

Recipes

  1. Murashige and Skoog medium
    0.025 mg/L CoCl2·6H2O
    0.025 mg/L CuSO4·5H2O
    36.7 mg/L Na·Fe-EDTA
    6.2 mg/L H3BO3
    0.83 mg/L KI
    16.9 mg/L MnSO4·2H2O
    0.25 mg/L Na2MoO4·2H2O
    8.6 mg/L ZnSO4·7H2O
    332.02 mg/L CaCl2·2H2O
    170 mg/L KH2PO4
    1,900 mg/L KNO3
    180.5 mg/L MgSO4·7H2O
    1,650 mg/L NH4NO3 (pH 5.8)
    1% sucrose
    0.5% phytagel
  2. Wash buffer
    10% formamide
    2x SSC
  3. Hybridization solution
    100 mg/ml dextran sulphate
    10% formamide
    2x SSC
  4. smFISH probe stock (25 μM)
    Stellaris 5 nmol oligo probe set
    200 μl Tris-EDTA buffer solution
  5. DAPI solution
    100 ng/μl DAPI
    10% formamide
    2x SSC
  6. Anti-fade GLOX buffer (minus enzymes)
    0.4% glucose
    10 nM Tris-HCl
    2x SSC
  7. Anti-fade GLOX buffer (containing enzymes)
    For a final volume of 102 μl, mix:
    100 μl anti-fade GLOX minus enzyme solution
    1 μl glucose oxidase
    1 μl bovine liver catalase suspension (mildy vortexed)

Acknowledgments

This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/K00008X/1 and the Earth and Life Systems Alliance (a collaborative venture between John Innes Centre and University of East Anglia). S.R. acknowledges support from 3.3-GRO/1162118STP from Humboldt Foundation (Germany). S.D. acknowledges support from OpenPlant Grant BB/L014130/1. C.D. acknowledges support from European Research Council Advanced grant MEXTIM and BBSRC Institute Strategic Programme grant BB/J004588/1. The original work was published in Duncan et al. (2016) and Rosa et al. (2016).

References

  1. Duncan, S., Olsson, T. S. G., Hartley, M., Dean, C. and Rosa, S. (2016). A method to detect single molecules of RNA in Arabidopsis thaliana. Plant Methods 12(1): 1-10
  2. Mueller, F., Senecal, A., Tantale, K., Marie-Nelly, H., Ly, N., Collin, O., Basyuk, E., Bertrand, E., Darzacq, X. and Zimmer, C. (2013). FISH-quant: automatic counting of transcripts in 3D FISH images. Nat Methods 10(4): 277-278.
  3. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. and Tyagi, S. (2008). Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10): 877-879.
  4. Rosa, S., Duncan, S. and Dean, C. (2016). Mutually exclusive sense – antisense transcription at FLC facilitates environmentally induced gene repression. Nat Commun 7: 13031.
  5. Schindelin, J., Rueden, C. T., Hiner, M. C. and Eliceiri, K. W. (2015). The ImageJ ecosystem: An open platform for biomedical image analysis. Mol Reprod Dev 82(7-8): 518-29.

简介

允许研究基因表达调控的方法不断前进。在这里,我们提出了一种能够检测植物根细胞中单个mRNA分子的原位杂交方案,从而允许mRNA在固定样品内的准确定量和定位(Duncan等人, ,2016; Rosa等人,2016)。这种单分子RNA荧光原位杂交(smFISH)使用多个单标记寡核苷酸探针结合靶RNA并产生可以使用宽场荧光显微镜检测的衍射限制信号。我们调整了该方法的最新版本,该方法使用48个荧光标记的DNA寡核苷酸(20个)与每个转录物的不同部分杂交(Raj等人,2008)。这种方法简单易行,具有很好的应用于任何遗传背景的优点。

虽然已经开发了单分子FISH来定量测量培养细胞,组织切片和整体无脊椎动物生物体的单细胞水平的mRNA,但该方法未被优化用于植物中的单细胞。由于植物组织的内源性自发荧光,植物中的荧光成像是相当有挑战性的。在这里,我们报告一种检测植物中单RNA分子的方法。我们描述在固定的拟南芥根瘤胃细胞内的单一转录物的检测和自动计数。该方法产生隔离的单元和单细胞层,其与红色和远红色染料一起使用可以最大化限制背景噪声的信噪比。

关键字:单个RNA分子, 荧光原位杂交, 基因表达, 拟南芥, 转录

材料和试剂

  1. 1.5 ml微量离心管
  2. 无菌Sterilin TM 10cm平方厘米用于植物生长培养基的培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:109)
  3. 22×22毫米1号玻璃盖玻片(VWR,目录号:631-0124)
  4. 聚-L-赖氨酸载玻片(Sigma-Aldrich,目录号:P0425)
  5. 剃刀刀片(Agar Scientific,目录号:AGT586)
  6. 密封胶膜(Bemis,目录号:PM992)
  7. 杂交室
    注意:虽然这些产品可以在市场上销售(Corning,目录号:2551),我们使用了10厘米平方英尺的培养皿,外面涂上一层黑色绝缘胶带(RS Components,目录号:494-405)。然后将双层组织(KCWW,Kimberly-Clark,目录号:7557)置于碱中并用无菌水饱和,然后将载玻片置于单层Parafilm顶部(参见图1)。


    图1.用于smFISH实验的杂交室的图示

  8. 低成型制备盘(VWR,目录号:470144-866;或类似的边缘玻璃皿)
  9. 拟南芥根
  10. 次氯酸钠(NaClO)(VWR,BDH ,目录号:CABDH7038-4L)
  11. 蔗糖(Sigma-Aldrich,目录号:S9378-1KG)
  12. Phytagel(Sigma-Aldrich,目录号:P8169)
  13. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  14. 不含核酸酶的磷酸盐缓冲盐水溶液(PBS,10x)pH 7.4(Thermo Fisher Scientific,Ambion TM,目录号:AM9624)
  15. 液氮
  16. 适用于分子生物学的乙醇
  17. 无核酸酶20倍生理盐水 - 柠檬酸钠(20×SSC)(Thermo Fisher Scientific,Ambion TM,目录号:AM9763)
  18. 透明指甲油(电子显微镜科学,目录号:72180;或类似)
  19. 硫酸葡聚糖(Sigma-Aldrich,目录号:RES2029D)
  20. 定制RNA FISH Stellaris 探针套件
    我们使用在线Stellaris Probe Designer来设计我们的smFISH探针集: https://www.biosearchtech.com/support/education/stellaris-rna-fish 。我们使用默认屏蔽级别2来避免一般有问题的RNA序列,并选择最多48个探针,寡核苷酸长度为20 nt,探针之间的最小间距为2 nt。
    我们还执行了TAIR BLAST搜索( https://www.arabidopsis.org /Blast/index.jsp ),并综合考虑结果以确保目标特异性。我们发现Quasar 570和Quasar 670染料同样适合在拟南芥根瘤细胞中成像RNA,但是我们无法检测用荧光素修饰的探针标记的RNA。
  21. Murashige&具有维生素的Skoog基础培养基(任何等同的来源都是合适的)(PhytoTechnology Laboratories ®,目录号:M519)
  22. 不含核酸酶的Tris-EDTA缓冲溶液(10mM Tris-HCl,1mM EDTA pH8)(Sigma-Aldrich,目录号:93283)
  23. DAPI(Thermo Fisher Scientific,Molecular Probes TM,目录号:D1306)
  24. 葡萄糖(Sigma-Aldrich,目录号:158968-500G)
  25. Tris HCl缓冲液1 M pH 8,无核酸酶(Thermo Fisher Scientific,Ambion TM,目录号:AM9855G)
  26. 葡萄糖氧化酶(Sigma-Aldrich,目录号:G0543)
  27. 牛肝过氧化氢酶(Sigma-Aldrich,目录号:C3155)
  28. 去离子甲酰胺(Sigma-Aldrich,目录号:F9037)
  29. 1N HCl(Sigma-Aldrich,目录号:71763)
  30. 10%(v/v)漂白剂(Vortex 5-10%次氯酸钠,Procter& Gamble,UK)稀释于dH2 O
  31. TE缓冲液(10nM Tris-HCl,1mM EDTA,pH8.0)
  32. 无核酸酶水 - 未经DEPC处理(QIAGEN,目录号:129117)
  33. Murashige和Skoog媒体(见食谱)
  34. 洗涤缓冲液(见配方)
  35. 杂交解决方案(见配方)
  36. DAPI解决方案(见配方)
  37. 抗褪色GLOX缓冲液(减去酶)(参见食谱)
  38. 抗褪色GLOX缓冲液(含酶)(见配方)

设备

  1. 镊子(Fisher Scientific,S Murray TM ,目录号:E017/01;或类似的)
  2. Coplin罐(Sigma-Aldrich,目录号:S6016;或类似物)
  3. 植物生长室(Panasonic Healthcare,Sanyo,目录号:MLR-352H-PE;或类似物)
  4. 烟气柜(Labconco,目录号:2247300;或类似)
  5. 层流流动柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraguard TM ECO Clean Bench Floor Stand,目录号:50109313;或类似的)
  6. 实验室烤箱(Cole-Palmer,目录号:WZ-52412-83;或类似)
  7. 轨道摇床(Cole-Palmer,目录号:WZ-51700-13;或类似的)
  8. 宽场荧光显微镜
    注意:我们使用了Andor iXon EM-CCD摄像头(Andor,型号: iXon EMCCD相机),但是也使用针对低速优化的标准CCD相机获得了类似的图像光级成像。
  9. 高数值孔径(> 1.3)和60或100x油浸物镜
  10. 强光源,如汞或金属卤化物灯(氙气或LED通常不够亮)
  11. 适用于荧光团的滤光片

软件

  1. ImageJ软件: http://rsbweb.nih.gov/ij /index.html (Schindelin等,2015)
  2. FISHcount: https://github.com/JIC-CSB/FISHcount
  3. Graphpad Prism软件或MS Excel

程序

  1. 植物生长和显微镜载玻片制备
    1. 在1.5 ml微量离心管中倒置5%(v/v)次氯酸钠表面灭菌5 min,然后在无菌蒸馏水中冲洗3次。
    2. 将种子放在补充有1%(w/v)蔗糖和0.5%(w/v)植物细胞的Murashige和Skoog培养基(pH 5.8)上。
    3. 通过在黑暗中4℃下孵育2天来分层种子。然后,将幼苗在22±1℃,垂直取向的培养皿中生长至少5天(16小时光照和8小时黑暗)。

  2. 拟南芥的固定和制备(见图2的概述)


    图2.拟南芥根细胞中smFISH的方案步骤。 有关详细信息,请参阅主要文本。
     
    1. 将来自拟南芥幼苗(约1cm)的根尖取出,同时仍然在平板上并放入含有新鲜制备的4%多聚甲醛的小玻璃皿中,并在室温下在通风橱下孵育30分钟。 br />
    2. 从固定剂中取出根,用1x PBS洗两次。
    3. 将3-4根根部放在幻灯片上,盖上盖玻片。用拇指轻轻地将每根根茎放在载玻片上,小心避免破坏盖玻片。目的是充分发挥根以在单个细胞层中产生多个文件和分离的细胞(参见图3)。


      图3.低倍数图像根分生组织南瓜。用DAPI(蓝色)染色的细胞核。该方法产生许多单独的单元格,或单独的文件或文件。比例尺= 200μm
    4. 使用镊子将压扁的根部保持在盖玻片下方,并将每个载玻片浸入液氮中约5秒钟。从氮气中取出后,可以使盖玻片和载玻片之间的剃刀刀片松开,然后将盖玻片滑开。
    5. 将样品在室温下空气干燥至少30分钟。
      注意:为避免增加自身荧光水平,请勿将其置于干燥时间超过2小时。
    6. 通过将载玻片浸入含有70%乙醇的共同浴缸中至室温下至少1小时来使样品透过。固定根可以在杂交前一周内在2-8℃下储存在70%乙醇中。

  3. 探针杂交
    1. 从乙醇中取出载玻片,使残留的乙醇在室温下蒸发
    2. 用洗涤缓冲液在载玻片上洗涤2次5分钟。
    3. 通过向100μl杂交溶液(250nM终浓度)中加入1μl所需的探针储备溶液来制备探针溶液。
    4. 在每个载玻片上加入100μl探针溶液,用盖玻片盖住,以防止蒸发,并在阴凉处37°C的潮湿室中孵育至少4 h(或过夜)。

  4. 样品安装
    1. 取出盖子,并将样品在200μl洗涤缓冲液中洗涤2次,载玻片上5分钟
    2. 将载玻片浸入含有约30ml洗涤缓冲液的同种罐中,并在37℃,黑暗中孵育30分钟。
    3. 从容器中取出载玻片并加入100μlDAPI溶液,然后在黑暗中37℃孵育30分钟。
    4. 用移液器小心吸取DAPI溶液,并用〜100μl2x SSC冲洗。
    5. 取出2x SSC,加入100μl抗褪色GLOX缓冲液(减去酶)。让它平衡1-2分钟。
    6. 去除抗褪色GLOX缓冲液(减去酶),并在每个载玻片上加入100μl抗褪色GLOX缓冲液(含酶)。
    7. 用盖玻片覆盖样品,除去多余的抗褪色GLOX缓冲液(加酶)并用指甲油密封。
    8. 在同一天拍摄幻灯片,以避免样品干燥和荧光团褪色。

  5. 成像
    1. 图像采集在宽视场荧光显微镜上,每个z平面的光学切片尺寸为0.2μm,横跨细胞的整个体积(约5μm,取决于细胞大小)。曝光时间取决于显微镜设置(光源,目标,等)。我们使用蔡司Elyra PS采用100x油浸物镜(1.46 NA)和冷却电子倍增CCD(电荷耦合器件)Andor iXon 897相机(512 x 512,QE> 90%)拍摄了我们的图像。对于用Quasar 570标记的探针,使用561nm的激发线,并在570-640nm检测到信号;对于用Quasar 670标记的探针,642nm的激发线和在655-710nm处检测到信号;对于DAPI,405nm的激发线和在420-480nm的波长处检测到信号。通常,我们对DAPI使用30毫秒的曝光时间,对于Quasar染料使用200-300毫秒。
    2. 在图像采集后,可以使用ImageJ(Image/Stack/Z Project)中的最大投影命令将Z-stacks缩小为2D数据集。有关此步骤的详细说明,请参阅ImageJ用户指南: https://imagej.nih.gov/ij/docs/guide/user-guide.pdf 。最大投影命令将导致具有与单个RNA相对应的衍射受限点的图像。当外源探针与同一基因的内含子探针结合使用时,一些斑点将在核内共定位,对应于在固定时转录的新生RNA分子(图4)。


      图4.单细胞中FLC转录物的检测。 A.用于检测FLC转录物的探针的示意图:内含子(红色)和外来(绿色)。 FLC
      外显子以粗线表示,内含子显示为细线。 B.在拟南芥根瘤细胞中显示出新生RNA(红色)表达的mRNA(绿色)和 FLC 的代表性图像。细胞质中的衍射受限斑点对应于单个FLC mRNA分子(绿色),并且与内含子信号(红色)共定位的强点对应于转录位点。根据细胞周期阶段,内含子信号可以检测多达2个点(G1期)或4个点(G2期)。细胞核用DAPI染色,显示为蓝色。刻度棒=5μm。

数据分析

  1. 对于我们的图像分析,我们使用了公认的mRNA计数程序,该程序已被优化用于我们的实验设置。有关如何在Mac计算机上安装此程序的详细信息,请参见 https://github.com/JIC-CSB/FISHcount
    注意:对于希望在Windows机器上量化FISH数据的研究人员,我们建议使用FISH-quant(Mueller等,2013)。
  2. 图6显示了FISHcount产生的"RNA识别","细胞分割"和"每个细胞的mRNA"输出图像的例子。在对整个数据集执行分析之后,我们建议您手动检查每个"每个细胞的mRNA"输出图像(图6D),以确保图像分析工作流程通过不正确的分割未产生不准确的结果。图像分析工作流程的详细说明也可以在Duncan等人的"2016"(图5)中找到。
  3. 然后可以使用GraphPad Prism或MS Excel记录和绘制数据,如图7所示。


    图5.图像分析工作流程(改编自Duncan等,2016)


    图6.FLC mRNA的自动图像分析。 A.用FLC mRNA探针标记的细胞的代表性最大投影图像(绿色)。用DAPI标记的DNA(蓝色)。 B-D。显示用于确定阳性mRNA信号的顺序检测步骤的屏幕截图。 mRNA位点;细胞分裂; D.每个细胞的mRNA数量的输出图像。比例尺= 5微米。


    图7.每个细胞的FLC mRNA分子的频率分布从三个实验分析总共520个细胞。误差棒为±SEM。

食谱

  1. Murashige和Skoog媒体
    0.025mg/L CoCl 2·6H 2 O
    0.025mg/L CuSO 4·5H 2 O
    36.7 mg/L Na·Fe-EDTA
    6.2mg/L H 3 BO 3 3/
    0.83 mg/L KI
    16.9mg/L MnSO 4·2H 2 O ○
    0.25mg/L Na 2 MoO 4·2H 2 O
    8.6mg/L ZnSO 4·7H 2 O
    332.02mg/L CaCl 2·2H 2 O ○
    170mg/L KH PO 4
    1,900 mg/L KNO 3
    180.5mg/L MgSO 4·7H 2 O
    1,650mg/L NH 4 NO 3(pH 5.8)
    1%蔗糖
    0.5%phytagel
  2. 洗涤缓冲液
    10%甲酰胺
    2x SSC
  3. 杂交解决方案
    100毫克/毫升葡聚糖硫酸盐
    10%甲酰胺
    2x SSC
  4. smFISH探针库(25μM)
    Stellaris 5 nmol寡聚探针组
    200μlTris-EDTA缓冲溶液
  5. DAPI解决方案
    100 ng /μlDAPI
    10%甲酰胺
    2x SSC
  6. 抗褪色GLOX缓冲液(减酶)
    0.4%葡萄糖
    10nM Tris-HCl
    2x SSC
  7. 抗褪色GLOX缓冲液(含酶)
    最终体积为102μl,混合:
    100μl抗褪色GLOX减去酶溶液
    1μl葡萄糖氧化酶
    1μl牛肝过氧化氢酶悬浮液(温和涡旋)

致谢

这项工作得到英国生物科技和生物科学研究理事会(BBSRC)授予BB/K00008X/1和地球与生命系统联盟(John Innes Center与东英吉利大学合作)的支持。 S.R.承认来自洪堡基金会(德国)的3.3-GRO/1162118STP的支持。 S.D.承认OpenPlant Grant BB/L014130/1的支持。光盘。承认欧洲研究理事会高级授权MEXTIM和BBSRC研究所战略计划授权BB/J004588/1的支持。原始作品发表于Duncan等人。 (2016)和Rosa等人。 (2016)。

参考文献

  1. Duncan,S.,Olsson,TSG,Hartley,M.,Dean,C.andRosa,S。(2016)。检测拟南芥中单RNA分子的方法 em> Plant Methods 12(1):1-10
  2. Mueller,F.,Senecal,A.,Tantale,K.,Marie-Nelly,H.,Ly,N.,Collin,O.,Basyuk,E.,Bertrand,E.,Darzacq,X和Zimmer,C 。(2013)。 FISH-quant:自动计数3D FISH图像中的成绩单。 10(4):277-278。
  3. Raj,A.,van den Bogaard,P.,Rifkin,SA,van Oudenaarden,A.和Tyagi,S。(2008)。使用多个单独标记的探针成像个体mRNA分子。 Nat方法5(10):877-879。
  4. Rosa,S.,Duncan,S.and Dean,C.(2016)。  FLC 中的互斥义 - 反义转录促进环境诱导的基因抑制。 Nat Commun 7:13031.
  5. Schindelin,J.,Rueden,CT,Hiner,MC和Eliceiri,KW(2015)。  ImageJ生态系统:生物医学图像分析的开放平台。 Mol Reprod Dev 82(7-8):518-29。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Duncan, S., Olsson, T. S., Hartley, M., Dean, C. and Rosa, S. (2017). Single Molecule RNA FISH in Arabidopsis Root Cells. Bio-protocol 7(8): e2240. DOI: 10.21769/BioProtoc.2240.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。