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Single Molecule RNA FISH in the Mammalian Oocyte
哺乳动物卵母细胞中的单分子RNA荧光原位杂交(FISH)   

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Abstract

RNA fluorescence in situ hybridization is a method to localize and measure gene expression in individual cell or tissue. Using multiple specific fluorescently labeled oligonucleotides greatly increases signal-to-noise ratio and thus enables detection of single RNA molecule. Around forty different DNA oligonucleotides designed to common RNA target and labeled with single fluorophore at 3´ terminus hybridizes with target RNA in fixed cells. We adapt this method to visualize target RNA in the mammalian oocyte. The ability to detect single transcript in the mammalian oocyte was challenging due to its large cell size. This method consists of four simple steps: fixation, permeabilization, hybridization and imaging. The protocol is adapted to this large nonattached cell to visualize maternal RNAs.
Combination of various fluorophores allows detection of more RNA targets. This method might be used with organelle markers or expanded with immunofluorescence protocol.

Keywords: Oocyte(卵母细胞), Visualization RNA(可视化RNA), Fluorescent in situ hybridization(荧光原位杂交), Maternal RNA(产妇的RNA), Gene expression(基因的表达)

Materials and Reagents

  1. Cover glasses thickness No.1 22 x 22 mm (Marienfeld-Superior, catalog number: 0101050 )
  2. Fisherfinest Premium Microscope Slides (Thermo Fisher Scientific, catalog number: 125447 )
  3. Tissue culture 24-well plates (TPP Techno Plastic Products AG, catalog number: 92024 )
  4. Tissue culture 96-well plates (TPP Techno Plastic Products AG, catalog number: 92096 )
  5. Borosilicate glass capillary 3.3. (Hilgenberg GmbH, catalog number: 1409363 ), hand pipette for manipulation) with tip diameter around 100 µm (Hilgenberg GmbH)
  6. Microtube from Eppendorf 15 ml RNase free (Sigma-Aldrich, catalog number: 0030123.328 )
    Note: Pricing & availability is not currently available.
  7. Aluminum foil (29 cm x 150 m) (MAIS.s.r.o.)
  8. Oocytes from Six-weeks-old female CD1 mice; isolation of mouse oocytes described in Bio-protocol (Tetkova and Hancova, 2015), also (Susor et al., 2015)
  9. Paraformaldehyde final concentration 4% in PBS (store at -20 °C) (Thermo Fisher Scientific, Afla Aesar, catalog number: 30525894 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 30525894 ”.
  10. Triton X-100 final concentration 2% in 1x PBS (fresh) (Sigma-Aldrich, catalog number: 9002931 )
  11. 20x saline-sodium citrate (SSC) stock is diluted in RNase free water, in the same day of experiment (Sigma-Aldrich, catalog number: S6639 )
  12. Ethanol 100% (for final concentration 70% ethanol, freshly dilute 100% ethanol with DEPC water) (Merck Millipore Corporation, catalog number: 1085430250 )
  13. VECTASHIELD HardSetTM with DAPI (storage 2-8 °C) (Vector Laboratories, catalog number: H1500 )
  14. ProtectRNA RNase Inhibitor 500x concentrated (final working concentration is 1x; storage at 2-8 °C in dark) (Sigma-Aldrich, catalog number: R7397 )
  15. Stellaris probes (stock concentration 5 nmol) (Bioresearch Technologies)
  16. Nuclease-free water (Life Technologies, Ambion®, catalog number: AM9932 )
    Note: Currently, it is “Thermo Fisher Scientific, AmbionTM, catalog number: AM9932”.
  17. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417 )
  18. Poly(vinyl alcohol) (PVA) (Sigma-Aldrich, catalog number: 341584 )
  19. Formamide (5 ml for 10% final concentration) (Sigma-Aldrich, catalog number: F7503 )
  20. Dextran sulfate sodium salt from Leuconostoc spp. (Sigma-Aldrich, catalog number: D8906 )
  21. Wash buffer (see Recipes)
  22. Hybridization buffer (see Recipes)
  23. Isolation buffer PVA (see Recipes)

Equipment

  1. Stereomicroscope Zeiss 2000C (Thermo Fisher Scientific)
  2. Heating plate (P-lab)
  3. Multi Bio 3D mini-Shaker (Biosan Laboratories)
  4. Thermostat incubator mini I5110 (Labnet International)
  5. Confocal microscope Leica SP5 (Leica Microsystems)
  6. Filter sets appropriate for fluorophores and excitation lasers
  7. 40x or 63x or 100x oil immersion objective

Procedure

  1. Fixation and permeabilization of oocytes
    1. Wash isolated oocytes in 400 µl isolation buffer PVA and then transfer oocytes into 400 µl of 4% PFA for 25 min room temperature.
    2. Transfer oocytes into a 24-well dish with 500 µl/well of 2% Triton X-100 with 1x ProtectRNA RNase Inhibitor for 10 min at room temperature.
    3. Instead of step A2, permeabilize oocytes at room temperature with 70% ethanol for 20 min.
      Note: Perform hybridization directly after fixation.

  2. Preparing probe and hybridization
    Notes:
    1. For the first use of Stellaris probes, it is recommended to start 4 separate hybridization reactions by adding 1 μl each of 1:50, 1:100, 1:200, 1:400 working dilutions of probes to see which one is optimal. Negative control eGFP, no probe, Malat 1 (Figure 1).
    2. Warm hybridization buffer to room temperature and mix well. Spin 25 µM working stock probes and dilute to 100 nM 200x times to final concentration into hybridization buffer.
    1. 500 µl of 37 °C preheated wash buffer with 1x protect RNA RNase inhibitor used for rehydration cells for 15 min.
    2. Transfer oocytes into 96 well plate with 100 µl/per well hybridization buffer and add probe.
    3. Protect 96 well plate from light by aluminum foil, incubate in thermostat incubator overnight at 37 °C.
      Note: Shaking is not an essential step, it might be omitted.

  3. Post hybridization washes and mounting
    1. Wash 1 time in 150 µl 2x SSC for 5 min, shake 30 RPM and turning angle 360° at 30 sec of 96 well plate.
      Transfer oocytes into 96 well plate with 125 µl/per well of warm wash buffer. Incubate at least 50 min at 37 °C.
      Note: Shaking is not an essential step, it might be omitted.
    2. Rinse 1 time in 150 µl of 2x SSC for 2 min at room temperature.
    3. Put oocytes with 0.5 µl of SSC liquid on glass slide with 20 µl of Vectashiled-hardening mounting medium with DAPI, wait 1 min then cover with cover slip.

  4. Image sample
    1. Leave the slide in the dark chamber for 2 h at 4 °C.
    2. Next, scan with confocal laser scanning microscope.
    3. Take images in the same day to avoid sample drying and fluorophore fading.

  5. Imaging setup
    Typical settings for oocytes have two channels (red, blue) and objective HCX PL APO lambda 63.0 x 1.25 OIL UV. Another setup: For visualization of Cal flour 635 labeled probes used 633 laser the 618 nm excitation and emission 635 nm wavelength, laser power set at 10%, scan speed 100 Hz, format of image 512 x 512, pine hole 110 μm, Z-stack thickness: 10 μm and image analysis and merging by LAS AF.
    Note: To be able to compare images, make sure settings are identical between different experiments (e.g., laser intensity, gain, offset, scan speed, etc.).

    Negative control for hybridization without probe.


    Negative control of experiment. Oocyte without eGFP mRNA (green).


    Specific signal of Malat1 (green) in cumulus cells in high probe concentration (1: 50). Oocyte shows high background.

    Figure 1. Optimization of FISH protocol


    Figure 2. Only oocyte express Dazl mRNA however cumulus cells shows no green signal after FISH against Dazl mRNA. Line marks the edge of the oocyte, DAPI (blue), scale bar 10 µm.


    Figure 3. GV stage of mouse oocyte with green signal for cMos mRNA. DAPI (blue), scale bar 10 µm.

Recipes

  1. Wash buffer (50 ml)
    20x SSC (5 ml)
    Formamide (5 ml for 10% final concentration)
    Nuclease-free water (to 50 ml final volume)
    Note: Divide volume into 5 ml aliquots, freeze and stored at -20 °C.
  2. Hybridization buffer (10 ml)
    Dextran sulfate (1 g)
    20x SSC (1 ml)
    Formamide (1 ml for 10% final concentration) RNAse free, stored at 2-8 °C
    Nuclease-free water (to 10 ml final volume)
    Note: Divide volume into 0.5 ml aliquots, freeze and stored at -20 °C.
  3. Isolation buffer PVA (50 ml)
    Phosphate buffer solution (50 ml final volume)
    PVA 2 mg
    Protect RNA RNase inhibitor 1x concentrate
    Note: Wash buffer with the same percentage of formamide as hybridization buffer.

Acknowledgments

This work was supported by research grants 1) Charles University in Prague, Faculty of Cell Biology-GAUK 243-227026 and 2) GACR 13-12291S. Some principles of the described protocol have been adapted from Singer Lab Protocol: Published 1998 and from Christian Lanctot Protocol. The original work was published in Susor et al. (2015).

References

  1. Bioresearch Technologies: Design and ordering single molecule FISH probe.
  2. Femino, A. M., Fay, F. S., Fogarty, K. and Singer, R. H. (1998). Visualization of single RNA transcripts in situ. Science 280(5363): 585-590.
  3. Flemr, M. and Svoboda, P. (2011). Ribonucleoprotein localization in mouse oocytes. Methods 53(2): 136-141.
  4. Susor, A., Jansova, D., Cerna, R., Danylevska, A., Anger, M., Toralova, T., Malik, R., Supolikova, J., Cook, M. S., Oh, J. S. and Kubelka, M. (2015). Temporal and spatial regulation of translation in the mammalian oocyte via the mTOR-eIF4F pathway. Nat Commun 6: 6078.

简介

RNA荧光原位杂交是定位和测量个体细胞或组织中的基因表达的方法。 使用多个特异性荧光标记的寡核苷酸大大增加信噪比,从而使得能够检测单个RNA分子。 大约四十个不同的DNA寡核苷酸设计为常见的RNA靶标,并在3'端用单个荧光团标记,与固定细胞中的靶RNA杂交。 我们适应这种方法可视化目标RNA在哺乳动物卵母细胞。 在哺乳动物卵母细胞中检测单个转录物的能力由于其大的细胞大小而具有挑战性。 该方法由四个简单的步骤组成:固定,预稳定化,杂交和成像。 该方案适应这种大的非附着细胞以显现母体RNA。
各种荧光团的组合允许检测更多的RNA靶标。 该方法可以与细胞器标记一起使用或用免疫荧光方案扩增。

关键字:卵母细胞, 可视化RNA, 荧光原位杂交, 产妇的RNA, 基因的表达

材料和试剂

  1. 保护玻璃厚度No.122×22mm(Marienfeld-Superior,目录号:0101050)
  2. Fisherfinest Premium显微镜载玻片(Thermo Fisher Scientific,目录号:125447)
  3. 组织培养24孔板(TPP Techno Plastic Products AG,??目录号:92024)
  4. 组织培养96孔板(TPP Techno Plastic Products AG,??目录号:92096)
  5. 硼硅酸盐玻璃毛细管3.3。 (Hilgenberg GmbH,目录号:1409363),尖端直径约100μm的手动移液管(Hilgenberg GmbH)
  6. 来自Eppendorf的15ml无RNA酶(Sigma-Aldrich,目录号:0030123.328)的微量管
    注意:可用性目前不可用。
  7. 铝箔(29cm×150μm)(MAIS.s.r.o.)
  8. 来自六周龄雌性CD1小鼠的卵母细胞;生物方案中描述的小鼠卵母细胞的分离(Tetkova和Hancova,2015),也是(Susor等人,2015)
  9. 多聚甲醛终浓度在PBS(储存在-20℃)中为4%(Thermo Fisher Scientific,Afla Aesar,目录号:30525894)
    注意:目前,它是"Sigma-Aldrich,目录号:30525894"。
  10. Triton X-100最终浓度2%在1×PBS(新鲜)(Sigma-Aldrich,目录号:9002931)中2%
  11. 在实验的同一天(Sigma-Aldrich,目录号:S6639)将20x盐水 - 柠檬酸钠(SSC)原液稀释于无RNA酶的水中
  12. 乙醇100%(用于终浓度70%乙醇,用DEPC水新鲜稀释100%乙醇)(Merck Millipore Corporation,目录号:1085430250)
  13. VECASHIELD HardSet TM TM/DAPI(储存2-8℃)(Vector Laboratories,目录号:H1500)
  14. Protectx RNA酶抑制剂500x浓缩(最终工作浓度为1x;在2-8℃下在黑暗中储存)(Sigma-Aldrich,目录号:R7397)
  15. Stellaris探针(库存浓度5nmol)(Bioresearch Technologies)
  16. 无核酸酶水(Life Technologies,Ambion ,目录号:AM9932)
    注意:目前,"Thermo Fisher Scientific,Ambion TM ,目录号:AM9932" />
  17. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P4417)
  18. 聚(乙烯醇)(PVA)(Sigma-Aldrich,目录号:341584)
  19. 甲酰胺(5ml,10%终浓度)(Sigma-Aldrich,目录号:F7503)
  20. 来自明串珠菌(Leuconostoc)的硫酸葡聚糖钠盐。 (Sigma-Aldrich,目录号:D8906)
  21. 洗涤缓冲液(见配方)
  22. 杂交缓冲液(参见配方)
  23. 隔离缓冲液PVA(参见配方)

设备

  1. 立体显微镜Zeiss 2000C(Thermo Fisher Scientific)
  2. 加热板(P-lab)
  3. Multi Bio 3D mini-Shaker(Biosan Laboratories)
  4. 恒温培养箱mini I5110(Labnet International)
  5. 共聚焦显微镜Leica SP5(Leica Microsystems)
  6. 适用于荧光团和激发激光器的滤光片组
  7. 40x或63x或100x油浸物镜

程序

  1. 卵母细胞的固定和透化
    1. 在400μl分离缓冲液PVA中洗涤分离的卵母细胞,然后将卵母细胞转移到400μl的4%PFA中25分钟室温。
    2. 将卵母细胞转移到24孔培养皿中,用500μl/孔的2%Triton X-100与1x ProtectRNA RNase抑制剂在室温下孵育10分钟
    3. 代替步骤A2,在室温下用70%乙醇渗透卵母细胞20分钟 注意:在固定后立即进行杂交。

  2. 准备探针和杂交
    注意:
    1. 首次使用Stellaris探头时,建议从4开始使用 通过加入1μl各1:50,1:100, 1:200,1:400工作稀释度的探针,以了解哪一种是最佳的。 阴性对照eGFP,无探针,Malat 1(图1)。
    2. 温暖 杂交缓冲液至室温并混匀。自旋25μM 工作母液探针稀释至100 nM 200x倍至最终 浓缩成杂交缓冲液。

    1. 500μl37℃预热的洗涤缓冲液,含1×保护RNA RNA酶抑制剂,用于15分钟的补液细胞。
    2. 转移卵母细胞到96孔板用100μl/每孔杂交缓冲液,并添加探针
    3. 通过铝箔保护96孔板免受光照,在37℃的恒温培养箱中孵育过夜 注意:抖动不是必要步骤,可能会被省略。

  3. 杂交后洗涤和安装
    1. 在150μl2x SSC中洗涤1次,每次5分钟,摇动30RPM,在96孔板上以30秒转动角度360°。
      转移卵母细胞96孔板用125微升/每孔的温水洗涤缓冲液。在37℃下孵育至少50分钟。
      注意:震动不是必要步骤,可能会被省略。
    2. 在室温下,在150μl的2x SSC中冲洗1次,每次2分钟
    3. 将卵母细胞与0.5微升的SSC液体在载玻片上与20微升 Vectashiled硬化封固剂用DAPI,等待1分钟,然后盖 与盖玻片。

  4. 图像样本
    1. 离开幻灯片在暗室在4℃下2小时。
    2. 接下来,用共焦激光扫描显微镜扫描。
    3. 在同一天拍摄图像,以避免样品干燥和荧光衰退。

  5. 成像设置
    卵母细胞的典型设置有两个通道(红色,蓝色)和客观HCX PL APO lambda 63.0 x 1.25 OIL UV。另一个设置:为了可视化Cal面粉635标记探针使用633激光618 nm激发和发射635 nm波长,激光功率设置为10%,扫描速度100 Hz,图像格式512 x 512,松孔110μm,Z - 堆叠厚度:10μm,并通过LAS AF进行图像分析和合并 注意:为了能够比较图像,请确保不同实验之间的设置相同(例如激光强度,增益,偏移,扫描速度等)。

    阴性对照,无探针杂交

    实验阴性对照。卵母细胞没有 eGFP mRNA(绿色)

    在高探针浓度(1:50)的卵丘细胞中的 Malat1(绿色)的特异性信号。卵母细胞显示出高背景
    图1. FISH协议的优化


    图2.只有卵母细胞表达dazl mRNA,而卵丘细胞在针对dazl mRNA后的FISH后没有显示绿色信号。卵母细胞DAPI (蓝色),比例尺为10μm

    图3.小鼠卵母细胞的GV期与cMos mRNA的绿色信号。 DAPI(蓝色),比例尺为10μm。

食谱

  1. 洗涤缓冲液(50ml)
    20×SSC(5ml) 甲酰胺(5ml,10%终浓度) 无核酸酶水(至最终体积为50ml)
    注意:将体积分成5毫升等分试样,冷冻保存在-20°C。
  2. 杂交缓冲液(10ml)
    硫酸葡聚糖(1g)
    20x SSC(1ml) 甲酰胺(1ml,10%终浓度)无RNA酶,储存在2-8℃下 无核酸酶水(至10ml终体积)
    注意:将体积分成0.5ml等分试样,冷冻并储存在-20℃。
  3. 分离缓冲液PVA(50ml)
    磷酸盐缓冲液(50ml终体积) PVA 2mg
    保护RNA RNA酶抑制剂1x浓缩物
    注意:用与杂交缓冲液相同百分比的甲酰胺冲洗缓冲液。

致谢

这项工作是由研究资助1)布拉格的查尔斯大学,细胞生物学学院 - GAUK 243-227026和2)GACR 13-12291S支持。所描述的协议的一些原理已经改编自Singer Lab Protocol:Published 1998和Christian Lanctot Protocol。原始的作品发表在Susor等人(2015)。

参考文献

  1. 生物研究技术:设计和订购单分子FISH探针。
  2. Femino,A.M.,Fay,F.S.,Fogarty,K。和Singer,R.H。(1998)。 原位显示单个RNA转录物 。 > Science 280(5363):585-590。
  3. Flemr,M。和Svoboda,P。(2011)。 小鼠卵母细胞中的核糖核蛋白定位。方法 53(2 ):136-141。
  4. Susor,A.,Jansova,D.,Cerna,R.,Danylevska,A.,Anger,M.,Toralova,T.,Malik,R.,Supolikova,J.,Cook,MS,Oh,JSand Kubelka, (2015年)。 通过mTOR-eIF4F通路在哺乳动物卵母细胞中翻译的时空和空间调节。 Nat Commun 6:6078.
  • 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: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jansova, D. (2015). Single Molecule RNA FISH in the Mammalian Oocyte. Bio-protocol 5(23): e1666. DOI: 10.21769/BioProtoc.1666.
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