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Metabolic Labeling of Yeast RNA with Radioactive Uracil
同位素示踪法进行酵母RNA的代谢标记   

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Abstract

To examine gene expression, Northern blot or Real-Time PCR can be used to detect low abundant RNA such as mRNA. However, for high abundant RNAs such as rRNA and tRNA, Northern blot will not be able to discriminate the newly synthesized RNA from total RNA. Therefore, metabolic labeling is necessary to evaluate the expression of rRNA and tRNA genes. In this protocol, I describe a step-by-step method for labeling yeast RNA with radioactive uracil and examine the synthesis of these high abundant RNAs.

Keywords: Autoradiography(放射自显影), Metabolic labeling(代谢标记), Tritium(氚), RRNA(rRNA), TRNA(tRNA)

Materials and Reagents

  1. Yeast strain of interest
  2. RapidGel (500 ml) (Affymetrix, catalog number: 75848 )
  3. Urea (CO(NH2)2) (Sigma-Aldrich, catalog number: U6504 )
  4. Dextrose (C6H12O6) (Sigma-Aldrich, catalog number: G7021 )
  5. Yeast nitrogen base without amino acids (Sigma-Aldrich, catalog number: Y0626 )
  6. Synthetic dropout supplement without uracil (Sigma-Aldrich, catalog number: Y1501 )
  7. Uracil (C4H4N2O2) (Sigma-Aldrich, catalog number: U1128 )
  8. Rapamycin
  9. TEMED/Tetramethylethylenediamine (C6H16N2) (Thermo Fisher Scientific, catalog number: 110-18-9 )
  10. Ammonium persulfate (APS) ((NH4)2S2O8) (Sigma-Aldrich, catalog number: A3678 )
  11. Formamide (CH3NO) (Thermo Fisher Scientific, catalog number: 75-12-7 )
  12. DEPC/ Diethylpyrocarbonate (O(COOC2H5)2) (Sigma-Aldrich, catalog number: D5758 )
  13. Bromophenol Blue (C19H10Br4O5S) (Sigma-Aldrich, catalog number: B0126 )
  14. Xylene Cyanol FF (C25H27N2NaO6S2) (Sigma-Aldrich, catalog number: X4126 )
  15. [5, 6-3H]-Uracil (PerkinElmer, catalog number: NET368250UC )
  16. RNA marker (Promega Corporation, catalog number: G3191 )
  17. Tris base (Thermo Fisher Scientific, catalog number: 77-86-1 )
  18. Boric acid (Thermo Fisher Scientific, catalog number: 10043-35-3 )
  19. EDTA (Sigma-Aldrich, catalog number: EDS-1KG )
  20. Formamide loading dye
  21. Small RNA separating gel
  22. SD-Ura- (see Recipes)
  23. SD-1/3 Uracil (see Recipes)
  24. DEPC water (see Recipes)
  25. 10x TBE in DEPC water (see Recipes)
  26. Small RNA separating gel (see Recipes)
  27. 2x Formamide loading dye (see Recipes)

Equipment

  1. Bench top centrifuge
  2. 30 °C Shaker
  3. Power supply with constant voltage > 450 V
  4. Gel dryer (Bio-Rad Laboratories, catalog number: 165-1745 )
  5. Exposure cosset/intensifier screen (Sigma-Aldrich, catalog number: C5479-1EA )
  6. 50 ml conical tubes
  7. 1.5 ml Eppendorf tube

Procedure

  1. Inoculate yeast single colony in SD medium (or SD with appropriate dropouts). Shake 300 rpm at 30 °C overnight.
  2. Dilute overnight culture in 50 ml conical tubes to 10 ml, OD600 =0.1 with SD-1/3 uracil and continue shaking until OD600 =0.4 (note: Reduction in cold uracil will allow hot uracil to be taken up by cells easily).
  3. Cells were treated with drug and control vehicle, for example 100 nM rapamycin (final concentration) and its solvent methanol, continue shaking 300 rpm at 30 °C for desired time. In this specific experiment, rapamycin were added for 30 min.
  4. Collect yeast cells by spinning down at 1,000 x g for 1 min at room temperature, remove supernatant (critical: Avoid putting yeast cells on ice. This is because ice will slow down growth, which will reduce significantly the uptake of hot uracil in the step 8 below).
  5. Re-suspend yeast cells in 1 ml SD-Ura- (critical: Pre-warm medium to 30 °C), transfer to 1.5 ml eppendorf tube.
  6. Spin down briefly by a bench top centrifuge at 5,000 x g, 15 sec, remove supernatant. Re-suspend yeast cells in 1 ml pre-warmed SD-Ura-.
  7. Repeat 6 for 2 times and with the final re-suspension in 0.5 ml pre-warmed SD-Ura- (from the next step, collect radioactive liquid and solid waste in all steps, dispose according to environmental regulation).
  8. Carefully add [5, 6-3H]-Uracil into each tube to the final concentration of 15 μCi/ml, vortex to mix, then put on a rack at 30 °C for 5 min.
  9. Briefly spin down by a bench top centrifuge at 5,000 x g, 15 sec, remove supernatant.
  10. Wash cells with SD-Ura-3 times as in step 5, ready to extract total RNA.
  11. Total RNA was extracted by hot phenol method described in Wei (2012).
  12. RNAs can be stored at -80 °C for up to 6 months.
  13. Prepare ''small RNA separating gel'' on a large gel set (around 20 x 30 cm, mini gel did not work well).
  14. Pre-run the gel for about 1 h at constant 450 V until the gel is heated to 50 °C.
    Note: I found this step to be critical. One reason could be that pre-running the gel to this temperature could help get rid of excessive Urea in the gel, making RNA possible to go through. I usually attached a thermometer to ensure that the temperature has reached 50 °C.
  15. Mix RNA samples with ''2x Formamide loading dye'' and heat at 70 °C for 2 min, then put samples on ice.
    Turn off power. Rinse the wells with 1x TBE using a syringe with needle, make sure all urea is rinsed out from the wells.
    Note: Urea is very dense and it will be impossible to load samples if urea is not rinsed out. If residual urea remains in the well, the resulting bands will be waving.
  16. Load RNA samples (25 μg) with appropriate RNA marker and run gel at constant 450 V for about 2 h (BPB runs around 12 nt and cyanol around 55 nt).
  17. Stain the gel with EtBr and take picture under UV light. This is total RNA which served as controls for newly synthesized RNA.
  18. Sandwich the gel with autoclaved filter paper on one side and Saran-Wrap on the other side, put on a gel-dryer with filter paper side attach to the vacuum surface. Dry the gel at 80 °C for at least 2 h. Sometimes gels get cracked, may be because of insufficient drying or leaky vacuum.
  19. The dried gel will stick to the filter paper. Wrap them in Sara-Wrap. 3H autoradiography in an exposure cassette with appropriate intensifying screen, for example Sigma Transcreen.
  20. Develop after 4-7 days of exposure.

Recipes

  1. SD-Ura-
    Synthetic dextrose medium with Uracil dropout:
    20 g Dextrose
    1.7 g Yeast Nitrogen Base
    1.92 g synthetic dropout supplement without Uracil
    5.0 g Ammonium Sulfate
    Add ddH2O2 to 1 L and autoclave.
  2. SD-1/3 Uracil: SD medium with 1/3 of regular Uracil
    Similar to SD-Ura-except adding 25 mg Uracil.
  3. DEPC water
    Add 1 ml DEPC to 1 L ddH2O2, mix and put at room temperature overnight. Autoclave.
  4. 10x TBE in DEPC water
    In 800 ml DEPC water, add:
    108 g Tris base
    55 g boric acid
    40 ml of 0.5 M EDTA (pH 8.0)
    Mix to dissolve and add DEPC water to 1 L. Autoclave.
  5. Small RNA separating gel
    1. Mix 2.5 ml 10x TBE, 6.25 ml RapidGel (40%) and 15 g Urea, heat to 50 °C and mix to dissolve.
    2. Add DEPC water to 25 ml then filter through 0.45 μM Syringe.
    3. Add 25 μl TEMED and 50 μl 25% APS, mix vigorously transfer to gel set with appropriate comb.
  6. 2x Formamide loading dye
    95% (v/v) formamide in DEPC water, add tiny amount of Bromophenol Blue (0.01~0.1%) and Xylene Cyanol FF (0.01~0.1%), vortex to mix.

Acknowledgments

This protocol is derived from the following papers, Wei et al. (2009a) and Wei et al. (2009b), and the relevant references therein. The work was supported by NIH grants R01-CA099004 and R01-CA123391 to Dr. X.F. Steven Zheng at Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey.

References

  1. Wei, Y., Tsang, C. K. and Zheng, X. F. (2009a). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28 (15): 2220-2230.
  2. Wei, Y. and Zheng, X. F. (2009b). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090.
  3. Wei, Y. (2012). A Simple Preparation of RNA from Yeast by Hot Phenol for Northern Blot. Bio-protocol 2(9): e209. 

简介

为了检查基因表达,可以使用Northern印迹或实时PCR来检测低丰度RNA如mRNA。 然而,对于高丰度的RNA如rRNA和tRNA,Northern印迹将不能区分新合成的RNA与总RNA。 因此,代谢标记是必要的,以评估rRNA和tRNA基因的表达。 在这个协议,我描述了一个分步的方法,用放射性尿嘧啶标记酵母RNA,并检查这些高丰度RNA的合成。

关键字:放射自显影, 代谢标记, 氚, rRNA, tRNA

材料和试剂

  1. 感兴趣的酵母菌株
  2. RapidGel(500ml)(Affymetrix,目录号:75848)
  3. 尿素(CO(NH 2)2)(Sigma-Aldrich,目录号:U6504)
  4. 葡萄糖(C 6 H 12 SO 6)(Sigma-Aldrich,目录号:G7021)
  5. 无氨基酸的酵母氮源(Sigma-Aldrich,目录号:Y0626)
  6. 不含尿嘧啶的合成辍学补充剂(Sigma-Aldrich,目录号:Y1501)
  7. 尿嘧啶(C 4 H 4 N 2 O 2 O 2)(Sigma-Aldrich,目录号:U1128) br />
  8. 雷帕霉素
  9. TEMED /四甲基乙二胺(C 6 H 16 N 12)(Thermo Fisher Scientific,目录号:110-18-9)
  10. 将过硫酸铵(APS)((NH 4)2 SS 2 O 8)(Sigma-Aldrich,目录 号码:A3678)
  11. 甲酰胺(CH 3 NO)(Thermo Fisher Scientific,目录号:75-12-7)
  12. DEPC /焦碳酸二乙酯(O(COOC H 2 H 5)2)(Sigma-Aldrich,目录号:D5758)
  13. 溴代酚蓝(C 19 H 10 BrB 4 O 5 S)(Sigma-Aldrich,目录号:B0126 )
  14. 二甲苯Cyanol FF(C 25 H 27 N sub 2 NaO 6 S 2)(Sigma-Aldrich,目录号:X4126 )
  15. [5,6-] 3 H] - 尿嘧啶(PerkinElmer,目录号:NET368250UC)
  16. RNA标记(Promega Corporation,目录号:G3191)
  17. Tris碱(Thermo Fisher Scientific,目录号:77-86-1)
  18. 硼酸(Thermo Fisher Scientific,目录号:10043-35-3)
  19. EDTA(Sigma-Aldrich,目录号:EDS-1KG)
  20. 甲酰胺负载染料
  21. 小RNA分离凝胶
  22. SD-Ura-(见配方)
  23. SD-1/3尿嘧啶(见配方)
  24. DEPC水(见配方)
  25. 在DEPC水中10x TBE(见配方)
  26. 小RNA分离凝胶(参见配方)
  27. 2x甲酰胺负载染料(参见配方)

设备

  1. 台式离心机
  2. 30℃摇床
  3. 恒压电源> 450 V
  4. 凝胶干燥器(Bio-Rad Laboratories,目录号:165-1745)
  5. 曝光cosset /增强屏(Sigma-Aldrich,目录号:C5479-1EA)
  6. 50ml锥形管
  7. 1.5 ml Eppendorf管

程序

  1. 接种酵母单个菌落在SD培养基(或SD与适当的失落)。在30℃下以300rpm振荡过夜。
  2. 使用SD-1/3尿嘧啶将50ml锥形管中的过夜培养物稀释至10ml,OD 600 = 0.1,并继续摇动直至OD 600 = 0.4(注意:冷尿嘧啶的减少将使得热尿嘧啶容易被细胞摄取。)
  3. 用药物和对照载体(例如100nM雷帕霉素(最终浓度))及其溶剂甲醇处理细胞,在30℃下继续振荡300rpm达所需时间。在该具体实验中,加入雷帕霉素30分钟
  4. 通过在室温下以1,000×g离心1分钟收集酵母细胞,除去上清液(关键:避免将酵母细胞置于冰上,这是因为冰会减缓生长,这将减少显着地在下面的步骤8中吸收热尿嘧啶)。
  5. 将酵母细胞重悬在1ml SD-Ura-(临界:预温培养基至30℃)中,转移至1.5ml eppendorf管中。
  6. 通过台式离心机在5000×g下短暂旋转15秒,除去上清液。 重新悬浮酵母细胞在1ml预热的SD-Ura-。
  7. 重复6次2次,最后再悬浮于0.5ml预热的SD-Ura-(从下一步,在所有步骤收集放射性液体和固体废物,根据环境规定处置)。
  8. 小心地将[5,6-,3 H] - 尿嘧啶添加到每个管中,使最终浓度为15μCi/ml,涡旋混合,然后在30℃放在架子上5分钟。
  9. 用台式离心机在5000×g下简单旋转15秒,除去上清液。
  10. 洗涤细胞与SD-Ura-3次,如步骤5,准备提取总RNA。
  11. 总RNA通过Wei(2012)中描述的热酚法提取
  12. RNA可以在-80℃保存长达6个月
  13. 在大凝胶组(约20×30cm,微型凝胶不能很好地)上制备"小RNA分离凝胶"。
  14. 预先运行凝胶约1小时,在恒定的450伏直到凝胶被加热到50℃。
    注意:我发现这一步很重要。一个原因可能是将凝胶预先运行到这个温度可以帮助除去凝胶中过多的尿素,使得RNA可能通过。我通常安装一个温度计,以确保温度已达到50°C。
  15. 将RNA样品与"2x甲酰胺负载染料"混合,在70℃加热2分钟,然后将样品置于冰上。
    关闭电源。使用带针头的注射器用1x TBE冲洗孔,确保所有尿素从孔中冲洗出来。
    注意:尿素非常稠密,如果尿素未被冲洗,将不可能装载样品。如果残留的尿素留在井中,则产生的带会波动。
  16. 加载RNA样品(25微克)与适当的RNA标记和运行凝胶在恒定的450伏约2小时(BPB运行约12 nt和cyanol约55 nt)。
  17. 用EtBr染色凝胶并在紫外光下拍照。 这是用作新合成的RNA的对照的总RNA
  18. 在一侧用高压灭菌的滤纸夹住凝胶,在另一侧将Saran-Wrap夹在凝胶上,放在具有滤纸侧的凝胶干燥器上,连接到真空表面。 在80℃下干燥凝胶至少2小时。 有时凝胶会破裂,可能是因为干燥不充分或漏气真空
  19. 干燥的凝胶将粘附到滤纸上。 包装在Sara-Wrap。 在具有适当增强屏幕的曝光盒(例如Sigma Transcreen)中的 3放射自显影。
  20. 暴露后4-7天开发。

食谱

  1. SD-Ura-
    尿嘧啶缺失的合成葡萄糖培养基:
    20克葡萄糖
    1.7克酵母氮基团
    1.92克不含尿嘧啶的合成辍学补充剂
    5.0克硫酸铵
    将ddH 2加至1L并高压灭菌。
  2. SD-1/3尿嘧啶:具有1/3尿嘧啶常规尿嘧啶的SD培养基 类似于SD-Ura,除了添加25mg尿嘧啶
  3. DEPC水
    将1ml DEPC加入1L ddH 2 O 2中,混合并在室温下放置过夜。 高压灭菌。
  4. DEPC水中10x TBE
    在800ml DEPC水中,加入:
    108克Tris碱
    55克硼酸 40ml 0.5M EDTA(pH8.0)
    混合溶解并加DEPC水至1 L.高压灭菌。
  5. 小RNA分离凝胶
    1. 将2.5ml 10x TBE,6.25ml RapidGel(40%)和15g尿素混合,加热至50℃,混合至溶解。
    2. 加入DEPC水至25ml,然后通过0.45μM注射器过滤。
    3. 加入25微升TEMED和50微升25%APS,混合剧烈转移到凝胶设置与适当的梳。
  6. 2x甲酰胺负载染料
    95%(v/v)甲酰胺的DEPC水溶液中,加入微量的溴酚蓝(0.01〜0.1%)和二甲苯Cyanol FF(0.01〜0.1%

致谢

该协议衍生自以下论文:Wei等人(2009a)和Wei等人(2009b)及其中的相关参考文献。 该工作由NIH授予R01-CA099004和R01-CA123391支持给Dr. X.F. Steven Zheng在Robert Wood Johnson医学院,新泽西州医学和牙科大学。

参考文献

  1. Wei,Y.,Tsang,C.K。和Zheng,X.F。(2009a)。 TORC1调节RNA聚合酶III依赖性转录的机制。 EMBO J 28(15):2220-2230
  2. Wei,Y。和Zheng,X.F。(2009b)。 Sch9部分介导TORC1信号转导以控制核糖体RNA合成。 Cell Cycle 8(24):4085-4090。
  3. Wei,Y。(2012)。 通过热苯酚在Northern印迹中从酵母中简单制备RNA。 /em> 2(9):e209。
  • English
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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: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wei, Y. (2013). Metabolic Labeling of Yeast RNA with Radioactive Uracil. Bio-protocol 3(7): e623. DOI: 10.21769/BioProtoc.623.
  2. Wei, Y., Tsang, C. K. and Zheng, X. F. (2009a). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28 (15): 2220-2230.
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