参见作者原研究论文

本实验方案简略版
May 2015

本文章节


 

Single-cell Visualization of Chromosome Transcriptional Territories by RNA-paint
通过RNA染色显示单细胞的染色体转录区   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

We developed a FISH-based method to directly assess chromosome-wide transcriptional activity, thereby enabling the visualization of the actively transcribed fraction of a chromosome at the single-cell level. We applied this method to probe the activity of X-chromosomes and its instability in the context of human embryonic stem cells and cancer cells.

Keywords: Transcription (转录), Immuno-fluorescence (免疫荧光), Chromosome-wide (染色体范围)

Materials and Reagents

  1. 24-well plates (Sigma-Aldrich, catalog number: Z707791-126EA )
  2. 13 mm round coverslips (Thermo Fisher Scientific, catalog number: 174950 )
  3. Glass slides
  4. Filter (0.2 μm)
  5. Filtration unit (Merck Millipore, catalog number: SCGPS05RE )
  6. Human embryonic stem cells (H9, WIBR2 and HUES1) and cancer cells (TCCSUP and RT112)
  7. Matrigel
  8. PBS solution (Life Technologies)
  9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  10. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  11. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  12. PIPES (Sigma-Aldrich, catalog number: P6757 )
  13. NaOH
  14. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  15. Ribonucleoside vanadyl complex (New England Biolabs, catalog number: S1402 )
  16. Paraformaldehyde (PFA) (16%, EM grade) (VWR International, catalog number: 100503-916 )
  17. EtOH
  18. Human Cot-1 DNA (Life Technologies)
  19. Sodium acetate anhydrous (NaOAc) (Sigma-Aldrich, catalog number: W302406 )
  20. Deionized formamide (Sigma-Aldrich, catalog number: F9037 )
  21. Rubber cement
  22. 20x SSC (Sigma-Aldrich, catalog number: 93017 )
  23. 20 mg/ml BSA (New England Biolabs, catalog number: B9000S )
  24. Dextran sulfate (Sigma-Aldrich, catalog number: 67578 )
  25. Formamide (Sigma-Aldrich, catalog number: 47670 )
  26. Mounting medium (Vector Laboratories, catalog number: H-1200 )
  27. Cy3-labelled human X-Chromosome paint (Metasystem, catalog number: D-0323-050-OR )
  28. FITC-labelled human X-Chromosome paint (Metasystem, catalog number: D-0323-050-FI )
  29. CSK buffer (see Recipes)
  30. 3% PFA/PBS solution (see Recipes)
  31. Hybridisation buffer (2x) (see Recipes)
  32. Washing solution (50% formamide/2x SSC) (see Recipes)
  33. Denaturating solution (70% formamide/2x SSC) (see Recipes)

Equipment

  1. Hybridisation table (Boekel Scientific, model: 240000 )
  2. Shake 'N BakeTM hybridisation oven (Boekel Scientific, model: 136400 )
  3. Centrifuge (VWR, Ependorf®, model: 5417R )
  4. Millivac-mini vacuum pump (Merck Millipore, catalog number: XF5423050 )
  5. Ependorf® Thermomixer® R (Sigma-Aldrich, catalog number: T3317 )
    Note: This product has been discontinued.
  6. Fluorescent microscope (motorized stage) (Leica, model: DMI-6000 )

Procedure

  1. Prepare 24-well plates with 13 mm round coverslips at the bottom of the wells. Coat coverslips with matrigel for human embryonic stem cells. Split cells in colonies with a ratio of 1:5 on coverslips. Grow cells for 24 to 48 h.
  2. Wash cells with 1 ml PBS per well.
  3. Incubate cells for 5 min on ice with 0.5 ml per well of ice-cold CSK buffer freshly supplemented with 0.5% Triton X-100 and 2 mM vanadyl ribonucleoside complex. The core CSK buffer can be kept at 4 °C several months.
  4. Fix cells for 10 min at room temperature with 0.5 ml per well of PBS/3% PFA supplemented with 2 mM vanadyl ribonucleoside complex.
  5. Rinse cells with 1 ml per well 3 times with cold EtOH (70%) for 4 min.
  6. Either proceed to step 8 or the protocol can be stopped here by tightly wrapping the 24-well plate with parafilm, stretching it gently to ensure good fit and avoid evaporation. Cells can be stored at -20 °C for one month.
  7. On the day of the experiment, bring coverslips to 4 °C in 70% EtOH.
  8. For probe preparation, for each coverslip, add 290 µl of water to 5 µl of concentrated chromosome paint and 5 µl of human Cot-1 DNA (1 µl/µg). Precipitate DNA by adding 1/10 3 M NaOAc and 2.5 volumes of 100% EtOH and storing tubes overnight at -20 °C. Probes are prepared individually for each coverslip: 1 mix for 1 coverslip.
  9. Spin probes at 15,000 x g at 4 °C for 20 min. The pellet should be visible and is washed 2 times with 70% EtOH and spun for 10 min at 15,000 x g at 4 °C.
  10. Resuspend the pellet in 5 µl of 50% deionized formamide/50% hybridisation buffer (2x), and incubate for 10 min at 37 °C. 
  11. Denature the chromosome paint 7 min at 75 °C, incubate for 30 min at 37 °C and store it on ice.
  12. In the meantime, dehydrate coverslips in 90% and 100% EtOH for 4 min each. Coverslips are then air dried on Kimwipes, cells face up. Heat clean glass slides on the hybridisation table at 37 °C, where the moats have been prefilled with water.
  13. Dispense 5 µl of probe per coverslip on glass slides, and place coverslips on the probe, with cells facing down (Figure 1). Incubate the coverslips at 37 °C overnight, and evaporation around the coverslips is prevented using rubber cement to seal the coverslips to the glass slides.


    Figure 1. Manipulating coverslips. A. Putting 5 µl of probe on slides. B. Putting coverslips with cells face down on probe preparation.

  14. The next day, remove rubber cement by gently drawing it from the coverslips. Spill 2 ml of 2x SSC over the coverslips and glass slides, and gently start pushing around the coverslips by making them float in 2x SSC. Fully detached coverslips are transferred to 24-well plates for further washes. Wash coverslips 3 times with 1 ml per well of 50% formamide/2x SSC, and 3 times in 2x SSC, all washes at 42 °C. Incubate 24-well plates with coverslips in hybridization oven with low agitation (shaker speed set at medium speed).
  15. Mount coverslips on glass sides using 5 µl of mounting medium containing DAPI. Use transparent nail polish to seal the coverslips to the slides.
  16. Observe fluorescent signals with a fluorescent microscope (magnification 100x).
    1. For dual RNA-FISH involving chromosome paint (Vallot et al., 2015)
      1. RNA-FISH is performed in two-steps. First, RNA-FISH with chromosome paint is performed overnight as described above (steps 1-14). 
      2. Coverslips are washed 3-times in 50% formamide/2x SSC at 37 °C and re-incubated overnight with the second probe.
      3. Coverslips are washed 3-times in 50% formamide/2x SSC and 3-times in 2x SSC at 37 °C. Observe both signals simultaneously.
    2. For successive RNA/DNA-FISH with chromosome paint (Vallot et al., 2015)
      1. RNA-FISH is performed on slides as described above using a Cy3-labelled paint to visualize RNA, take pictures and note cell coordinates using a stage-motorized Leica microscope. 
      2. Wash slides, cells are permeabilized for 10 min in 0.1 N HCl/0.7% Triton X-100 on ice, treated with 100 µg/µl RNase in 2x SSC for 1 h at 37 °C. 
      3. Denature slides for 10 min at 80 °C in 70% formamide/2x SSC. 
      4. DNA is visualized using a FITC-labelled X chromosome paint, it is denatured for 2 min at 75 °C and incubated with slides overnight. 
      5. Wash slides 3 times with 2x SSC at 45 °C and 0.1x SSC at 60 °C.

Representative data



Figure 2. Optimizing the X-chromosome paint RNA FISH. 5 µl of cy3-X-chromosome paint are co-precipitated with various amounts of human Cot-1 DNA prior to over-night hybridisation. Optimization of the quantity of Cot-1 should be performed when changing supplier for chromosome paint.


Figure 3. Single-cell detection of X-chromosome activity in cancer cells. X-chromosome content of two bladder cancer cell lines RT112 (A) and TCCSUP (B) was assessed by DNA-FISH using FITC-labeled X-chromosome paint (left panel) and X-chromosome activity by RNA-paint using cy3-labeled X-chromosome paint (right panel).The percentage in the right lower corner indicates the percentage of nuclei with displayed pattern.

Recipes

  1. CSK buffer
    100 mM NaCl
    300 mM sucrose
    3 mM MgCl2
    10 mM PIPES
    Adjust pH to 6.8 with NaOH to facilitate dissolution of chemicals in water
    Filter (0.2 μm)
  2. 3% PFA/PBS solution
    9.4 ml of 16% PFA solution
    40.6 ml of PBS
  3. Hybridisation buffer (2x)
    4x SSC
    4 mg/ml BSA
    20% dextran sulfate
    40 mM vanadyl ribonuclside complex
  4. Washing solution (50% formamide/2x SSC) (50 ml), pH = 7.2
    25 ml of formamide
    5 ml of 20x SSC
    20 ml of H2O
    60 µl of 2.5 N HCl
  5. Denaturating solution (70% formamide/2x SSC) (50 ml), pH = 7.2
    35 ml of formamide
    5 ml of 20x SSC
    10 ml of H2O
    145 µl of 2.5 N HCl

Acknowledgments

We thank members of our laboratory for stimulating discussion. The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement No. 206875 and from the Inserm (Avenir Program R0721HS).

References

  1. Vallot, C., Ouimette, J. F., Makhlouf, M., Feraud, O., Pontis, J., Come, J., Martinat, C., Bennaceur-Griscelli, A., Lalande, M. and Rougeulle, C. (2015). Erosion of X chromosome inactivation in human pluripotent cells initiates with XACT coating and depends on a specific heterochromatin landscape. Cell Stem Cell 16(5): 533-546.

简介

我们开发了基于FISH的方法,以直接评估染色体宽转录活性,从而使单细胞水平的染色体的积极转录部分的可视化。 我们应用这种方法探测X染色体的活动及其在人类胚胎干细胞和癌细胞的上下文中的不稳定性。

关键字:转录, 免疫荧光, 染色体范围

材料和试剂

  1. 24孔板(Sigma-Aldrich,目录号:Z707791-126EA)
  2. 13mm圆形盖玻片(Thermo Fisher Scientific,目录号:174950)
  3. 玻璃滑槽
  4. 过滤器(0.2μm)
  5. 过滤单元(Merck Millipore,目录号:SCGPS05RE)
  6. 人胚胎干细胞(H9,WIBR2和HUES1)和癌细胞(TCCSUP和RT112)
  7. Matrigel
  8. PBS溶液(Life Technologies)
  9. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  10. 蔗糖(Sigma-Aldrich,目录号:SO389)
  11. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  12. PIPES(Sigma-Aldrich,目录号:P6757)
  13. NaOH
  14. Triton X-100(Sigma-Aldrich,目录号:X100)
  15. 核糖核苷氧钒基复合物(New England Biolabs,目录号:S1402)
  16. 多聚甲醛(PFA)(16%,EM级)(VWR International,目录号:100503-916)
  17. EtOH
  18. 人类Cot-1 DNA(Life Technologies)
  19. 无水乙酸钠(NaOAc)(Sigma-Aldrich,目录号:W302406)
  20. 去离子甲酰胺(Sigma-Aldrich,目录号:F9037)
  21. 橡胶水泥
  22. 20x SSC(Sigma-Aldrich,目录号:93017)
  23. 20 mg/ml BSA(New England Biolabs,目录号:B9000S)
  24. 硫酸葡聚糖(Sigma-Aldrich,目录号:67578)
  25. 甲酰胺(Sigma-Aldrich,目录号:47670)
  26. 安装介质(Vector Laboratories,目录号:H-1200)
  27. Cy3标记的人X染色体涂料(Metasystem,目录号:D-0323-050-OR)
  28. FITC标记的人X染色体涂料(Metasystem,目录号:D-0323-050-FI)
  29. CSK缓冲区(请参阅配方)
  30. 3%PFA/PBS溶液(参见配方)
  31. 杂交缓冲液(2x)(参见配方)
  32. 洗涤溶液(50%甲酰胺/2×SSC)(参见配方)
  33. 变性溶液(70%甲酰胺/2×SSC)(参见配方)

设备

  1. 杂交表(Boekel Scientific,型号:240000)
  2. Shake'N Bake TM 杂交炉(Boekel Scientific,型号:136400)
  3. 离心机(VWR,Ependorf ,型号:5417R)
  4. Millivac-mini真空泵(Merck Millipore,目录号:XF5423050)
  5. Ependorf Thermomixer R(Sigma-Aldrich,目录号:T3317)
    注意:此产品已停产。
  6. 荧光显微镜(机动载物台)(Leica,型号:DMI-6000)

程序

  1. 准备24孔板与13毫米圆盖玻片在底部的孔。用人胚胎干细胞的基质胶涂盖玻片。在盖玻片上以1:5的比例在菌落中分裂细胞。生长细胞24至48小时
  2. 用每孔1ml PBS洗涤细胞
  3. 在冰上孵育细胞5分钟,每孔0.5ml每孔新鲜补充0.5%Triton X-100和2mM氧钒核糖核苷复合物的冰冷的CSK缓冲液。核心CSK缓冲液可以在几个月内保持在4°C
  4. 修复细胞在室温下10分钟,用0.5毫升每孔的PBS/3%PFA补充2mM氧钒核糖核苷复合物。
  5. 用1ml /孔冲洗细胞3次,用冷的EtOH(70%)4分钟
  6. 可以进行到步骤8或者可以通过用石蜡膜紧紧包裹24孔板,轻轻地拉伸以确保良好的配合和避免蒸发来停止方案。细胞可以在-20℃保存一个月。
  7. 在实验当天,将盖玻片在70%EtOH中加热至4℃
  8. 对于探针制备,对于每个盖玻片,向5μl浓缩染色体涂料和5μl人Cot-1 DNA(1μl/μg)中加入290μl水。通过加入1/10 3 M NaOAc和2.5体积的100%EtOH沉淀DNA,并在-20℃下储存管过夜。每个盖玻片单独制备探针:1混合1盖玻片
  9. 在4℃下以15,000×g离心20分钟旋转探针。沉淀应该是可见的并且用70%EtOH洗涤2次并在4℃下以15,000×g离心10分钟。
  10. 将沉淀重悬于5μl50%去离子甲酰胺/50%杂交缓冲液(2x)中,并在37℃孵育10分钟。
  11. 在75°C将染色体涂料变性7分钟,在37°C孵育30分钟,并储存在冰上。
  12. 同时,脱水盖玻片在90%和100%乙醇中各4分钟。然后将盖玻片在Kimwipes上风干,细胞面朝上。在37℃下,在杂交台上热清洁玻璃载玻片,其中护城河已经 预装水。
  13. 每个盖玻片分配5微升探针在玻片上,并将盖玻片放置在探针上,细胞面朝下(图1)。孵育盖玻片在37℃过夜,并使用橡胶水泥密封盖玻片到玻璃载玻片防止盖玻片周围的蒸发。


    图1.操作盖玻片。A.将5μl探针放在载玻片上。 B.将具有细胞的盖玻片面向下放置在探针制备上
  14. 第二天,通过轻轻地从盖玻片上取下橡胶水泥。溢出2毫升2x SSC盖玻片和玻璃幻灯片,轻轻开始推动盖玻片,通过使它们在2x SSC浮动。完全分离的盖玻片被转移到24孔板用于进一步的洗涤。用1%每孔的50%甲酰胺/2×SSC洗涤盖玻片3次,在2x SSC中洗涤3次,全部在42℃洗涤。在具有低搅拌(振荡器速度设定为中等速度)的杂交烘箱中孵育具有盖玻片的24孔板。
  15. 使用5μl包含DAPI的封装介质在玻璃面上盖玻片。使用透明指甲油密封幻灯片上的盖玻片
  16. 用荧光显微镜观察荧光信号(放大100倍)。
    1. 对于涉及染色体涂层的双重RNA-FISH(Vallot等人,2015)
      1. RNA-FISH在两个步骤中进行。 首先,如上所述,用染色体涂料进行RNA-FISH过夜(步骤1-14)。
      2. 将盖玻片在37℃下在50%甲酰胺/2×SSC中洗涤3次,并用第二探针再温育过夜。
      3. 盖玻片在50%甲酰胺/2×SSC中洗涤3次,在2×SSC中在37℃下洗涤3次。 同时观察这两个信号。
    2. 对于具有染色体涂层的连续RNA/DNA-FISH(Vallot等人,2015)
      1. 如上所述使用Cy3标记的涂料在载玻片上进行RNA-FISH以显现RNA,使用阶段摩托化的徕卡显微镜拍摄照片和注意细胞坐标。
      2. 洗涤载玻片,将细胞在0.1N HCl/0.7%Triton X-100中在冰上透化10分钟,用100μg/μlRNA酶在2x SSC中在37℃处理1小时。< br />
      3. 变性在80℃在70%甲酰胺/2×SSC中滑动10分钟
      4. 使用FITC标记的X染色体涂料使DNA可视化,在75℃变性2分钟,并与载玻片一起温育。
      5. 用45℃的2x SSC和60℃的0.1×SSC洗涤载玻片3次。

代表数据



图2.优化X染色体RNA RNA FISH。在过夜杂交之前,将5μl的cy3-X染色体涂料与各种量的人Cot-1 DNA共沉淀。当更换染色体涂料的供应商时,应该优化Cot-1的数量

图3.癌细胞中X染色体活性的单细胞检测通过DNA-FISH使用FITC评估两个膀胱癌细胞系RT112(A)和TCCSUP(B)的X染色体含量标记的X染色体涂料(左图)和通过RNA涂料的X-染色体活性使用cy3标记的X-染色体涂料(右图)。右下角的百分比表示具有显示图案的细胞核的百分比。

食谱

  1. CSK缓冲区
    100 mM NaCl
    300mM蔗糖 3mM MgCl 2/
    10 mM PIPES
    用NaOH调节pH至6.8,以促进化学物质在水中溶解
    过滤器(0.2μm)
  2. 3%PFA/PBS溶液
    9.4ml 16%PFA溶液
    40.6ml PBS
  3. 杂交缓冲液(2x)
    4x SSC
    4mg/ml BSA
    20%硫酸葡聚糖
    40mM氧钒核糖核苷复合物
  4. 洗涤溶液(50%甲酰胺/2×SSC)(50ml),pH = 7.2 25ml甲酰胺 5ml 20×SSC
    20ml H 2 O 2 / 60μl2.5N HCl
  5. 变性溶液(70%甲酰胺/2×SSC)(50ml),pH = 7.2 35ml甲酰胺 5ml 20×SSC
    10ml H 2 O x/v 145μl2.5N HCl

致谢

我们感谢我们实验室的成员激发讨论。 导致这些结果的研究得到欧洲研究委员会根据欧洲共同体第七框架计划(FP7/2007-2013)/ERC赠款协议n°206875和Inserm(Avenir计划R0721HS)的资助。

参考文献

  1. Vallot,C.,Ouimette,JF,Makhlouf,M.,Feraud,O.,Pontis,J.,Come,J.,Martinat,C.,Bennaceur-Griscelli,A.,Lalande,M.and Rougeulle, (2015)。 人类多潜能细胞中X染色体失活的侵蚀起始于XACT涂层, 取决于特异性异染色质。细胞干细胞 16(5):533-546。
登录/注册账号可免费阅读全文
  • 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: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vallot, C. and Rougeulle, C. (2016). Single-cell Visualization of Chromosome Transcriptional Territories by RNA-paint. Bio-protocol 6(17): e1914. DOI: 10.21769/BioProtoc.1914.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。