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Compared to several expensive RNA extraction kits, the following protocol provides an economic and simple method for researchers to extract yeast RNA. This method can achieve RNA quality that is sufficient for most northern blot studies in yeast.

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A Simple Preparation of RNA from Yeast by Hot Phenol for Northern Blot
用于Northern印记杂交实验的酵母RNA的提取-简易热苯酚法

分子生物学 > RNA > RNA 提取
作者: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
Vol 2, Iss 12, 6/20/2012, 7625 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.209

[Abstract] Compared to several expensive RNA extraction kits, the following protocol provides an economic and simple method for researchers to extract yeast RNA. This method can achieve RNA quality that is sufficient for most northern blot studies in yeast.

[Abstract] 与几种昂贵的RNA提取试剂盒相比,以下方案为研究人员提取酵母RNA提供了经济和简单的方法。 这种方法可以实现RNA质量,足以用于酵母中的大多数Northern印迹研究。

Materials and Reagents

  1. W303a cell line
  2. Sodium acetate trihydrate (CH3CO2Na•3H2O) (Sigma-Aldrich, catalog number: 236500 )
  3. Phenol (C6H5OH) (Sigma-Aldrich, catalog number: P1037 )
  4. EDTA (Na2EDTA•2H2O) (Sigma-Aldrich, catalog number: ED2SS )
  5. Acetic acid (CH3COOH) (Sigma-Aldrich, catalog number: 320099 )
  6. Chloroform (CHCl3) (Sigma-Aldrich, catalog number: 472476 )
  7. 8-hydroxyquinoline (C9H7NO) (Sigma-Aldrich, catalog number: 252565 )
  8. NaCl (Thermo Fisher Scientific, catalog number: S641-500 )
  9. Synthetic complete (SC) medium
  10. SDS (Sigma-Aldrich, catalog number: L3771 )
  11. Isoamyl alcohol (Sigma-Aldrich, catalog number: W205710 )
  12. Ethanol (Thermo Fisher Scientific, catalog number: 64-17-5 )
  13. DEPC water
  14. Phenol-AE buffer (see Recipes)
  15. Phenol: CHCl3 /AE-Na (see Recipes)
  16. CHCl3: Isoamyl alcohol (24:1) (see Recipes)

Equipment

  1. Standard bench-top centrifuge
  2. Microfuge
  3. Shaker
  4. 1.5 eppendorf tube
  5. Liquid nitrogen

Procedure

  1. Prepare reagents according to recipes. Note that everything is in DEPC water.
  2. Inoculate W303a cells expressing different TOR1-RR variants in 2 ml SC medium overnight.
  3. Subculture the cells starting from OD600=0.1 in 10 ml SC media, shake vigorously at 30 °C, 300 rpm for around 4-6 h until OD600=0.4-0.5.
  4. Collect the cells by spinning down without freezing on ice. Discard supernatant.
  5. Re-suspend cells with 1 ml water and transfer to a 1.5 eppendorf tube, quickly spin down at 3,000 x g for 15 sec.
  6. Re-suspend cell pellet in 400 µl of AE buffer at room temperature.
  7. Add 40 µL 10% SDS (final around 1%) and vortex briefly at room temperature (RT).
  8. Immediately add 500 µl hot phenol/AE (put in 65 °C for 10 min before use), vortex vigorously for 1 min.
  9. Incubate at 65 °C for 5 min. Briefly vortex every 30 sec.
  10. Immediately freeze by dumping into liquid nitrogen (labels won’t get off).
  11. Wait to thaw at RT (put in 30 °C to thaw may crack the tube).
  12. Centrifuge for 10 min on a standard laboratory microfuge at 20,000 x g at RT.
  13. Transfer around 400 μl supernatant to a new eppendorf tube. Recycle the lower phenol fraction carefully following the chemical safety protocol in your laboratory.
  14. Add equal volume (400 μl) phenol: CHCl3/AE-Na. Vortex vigorously for 1 min at RT.
  15. Spin down at 20,000 x g for 5 min in a standard laboratory microfuge.
  16. Transfer supernatant (around 350 μl) to a fresh 1.5 ml eppendorf tube.
  17. Add CHCl3: isoamyl alcohol (24:1). Vortex vigorously for 1 min at RT.
  18. Transfer aqueous supernatant to fresh 1.5 ml microfuge tube. If white cloudy precipitate is observed between aqueous phase and organic phase, repeat steps 17-18.
  19. Add 1/10 volume of 3 M NaOAc (pH 5) and vortex vigorously. Add 2.5 volumes of ethanol. Vortex again.
  20. Place at -20 °C for at least 30 min.
  21. Spin down in the microfuge at 20,000 x g, 15 min at 4 °C. RNA pellet is usually visible.
  22. Add ice-cold 75% EtOH, place at 4 °C for around 10 min. Vortex and spin down on microfuge 20,000 x g, 15 min at 4 °C.
  23. Discard supernatant. Suck out the liquid droplets in the tube.
  24.  The white RNA pellet will turns clear when it dries out. Add 30-50 µl ddH2O (DEPC) immediately after it becomes clear.
  25. Do not let the RNA over-dry, which will make it difficult to dissolve. If RNA pellet is over-dry, dissolve RNA at 37 °C for 30 min.
  26. Store RNAs at -80 °C for more than 2 months.

Recipes

  1. Phenol-AE buffer
    Make Acetate-EDTA(AE) buffer
    50 mM NaOAc
    10 mM EDTA (pH 5.0)
    Liquefied phenol equilibrated with equal volume of AE buffer. Store at 4 °C.
  2. Phenol: CHCl3 /AE-Na
    Make AE-Na buffer
    10 mM NaOAc
    2 mM EDTA
    100 mM NaCl (pH 6.0)
    50% phenol/AE
    50% CHCl3
    0.25% 8-hydroxyquinoline
    Store at 4 °C.
  3. CHCl3: Isoamyl alcohol (24:1)

Acknowledgments

This protocol was adapted from and used in Wei and Zheng (2009) and Wei et al. (2009).

References

  1. Wei, Y. and Zheng, X. F. (2009). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090.
  2. Wei, Y., Tsang, C. K. and Zheng, X. F. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28(15): 2220-2230.

材料和试剂

  1. W303a细胞系
  2. 乙酸钠三水合物(CH 3 CO 2 Na 3·3H 2 O)(Sigma-Aldrich,目录号:236500)
  3. 苯酚(C 6 H 5 OH)(Sigma-Aldrich,目录号:P1037)
  4. EDTA(Na 2 EDTA; 2H 2 O)(Sigma-Aldrich,目录号:ED2SS)
  5. 乙酸(CH 3 COOH)(Sigma-Aldrich,目录号:320099)
  6. 氯仿(CHCl 3)(Sigma-Aldrich,目录号:472476)
  7. (C 9 H 7 NO)(Sigma-Aldrich,目录号:252565)。
  8. NaCl(Thermo Fisher Scientific,目录号:S641-500)
  9. 合成完全(SC)培养基
  10. SDS(Sigma-Aldrich,目录号:L3771)
  11. 异戊醇(Sigma-Aldrich,目录号:W205710)
  12. 乙醇(Thermo Fisher Scientific,目录号:64-17-5)
  13. DEPC水
  14. 苯酚AE缓冲液(参见配方)
  15. 苯酚:CHCl 3/AE-Na(参见配方)
  16. CHCl 3:异戊醇(24:1)(参见配方)

设备

  1. 标准台式离心机
  2. Microfuge
  3. 振动器
  4. 1.5 eppendorf管
  5. 液氮

程序

  1. 根据配方制备试剂。 注意,一切都在DEPC水中。
  2. 在2ml SC培养基中接种表达不同TOR1-RR变体的W303a细胞过夜
  3. 在10ml SC培养基中从OD 600 = 0.1开始继代培养细胞,在30℃,300rpm下剧烈摇动约4-6小时,直到OD 600 = 0.4- 0.5。
  4. 通过旋转收集细胞而不在冰上冷冻。 弃去上清液。
  5. 用1ml水重新悬浮细胞并转移到1.5微升管中,迅速离心3 000×g,15秒。
  6. 在室温下将细胞沉淀重悬在400μlAE缓冲液中
  7. 加入40μL10%SDS(最终约1%),并在室温(RT)下短暂涡旋
  8. 立即加入500μl热酚/AE(在使用前在65℃下放置10分钟),剧烈涡旋1分钟。
  9. 在65℃孵育5分钟。 简短涡旋每30秒。
  10. 立即通过倾倒到液氮中冷冻(标签不会脱落)。
  11. 等待在室温下解冻(放在30°C解冻可能会破裂管)
  12. 在标准实验室微量离心机上,在RT下以20,000×g离心10分钟
  13. 转移约400μl上清至新的eppendorf管。 按照您实验室中的化学安全方案,仔细回收低级苯酚馏分。
  14. 加入等体积(400μl)苯酚:CHCl 3/AE-Na。 在室温下剧烈涡旋1分钟
  15. 在标准实验室微量离心机中旋转5分钟,在20,000英尺×g
  16. 转移上清液(约350微升)到新鲜的1.5毫升eppendorf管
  17. 加入CHCl 3:异戊醇(24:1)。 在室温下剧烈涡旋1分钟
  18. 将含水上清液转移至新鲜的1.5ml微量离心管中。 如果在水相和有机相之间观察到白色浑浊的沉淀,重复步骤17-18
  19. 加入1/10体积的3M NaOAc(pH 5)并剧烈涡旋。 加入2.5倍体积的乙醇。 再次涡旋。
  20. 置于-20°C至少30分钟。
  21. 在微型离心机中以20,000×10 6细胞/分钟在4℃下旋转15分钟。 RNA沉淀通常是可见的
  22. 加入冰冷的75%EtOH,置于4℃约10分钟。 在4℃下涡旋并旋转离心20,000小时,15分钟。
  23. 弃去上清液。 吸出管中的液滴。
  24.  白色RNA颗粒在干燥时会变得清澈。 在澄清后立即加入30-50μlddH 2 O(DEPC)。
  25. 不要让RNA过干,这将使其难以溶解。 如果RNA沉淀过度干燥,溶解RNA在37℃下30分钟。
  26. 将RNA储存于-80℃超过2个月。

食谱

  1. 苯酚AE缓冲液
    制作乙酸盐-EDTA(AE)缓冲液
    50mM NaOAc
    10mM EDTA(pH5.0) 用等体积的AE缓冲液平衡的液化苯酚。 储存于4°C。
  2. 苯酚:CHCl 3/AE-Na
    生成AE-Na缓冲区
    10mM NaOAc
    2mM EDTA 100mM NaCl(pH 6.0)
    50%苯酚/AE
    50%CHCl 3
    0.25%8-羟基喹啉
    储存于4°C。
  3. CHCl 3:异戊醇(24:1)

致谢

该方案从Wei和Zheng(2009)和Wei等人(2009)改编并使用。

参考文献

  1. Wei,Y。和Zheng,X.F。(2009)。 Sch9 部分介导TORC1信号传导以控制核糖体RNA合成。 Cell Cycle 8(24):4085-4090。
  2. Wei,Y.,Tsang,C.K.and Zheng,X.F。(2009)。 TORC1调节RNA聚合酶III依赖性转录的机制 EMEM J 28(15):2220-2230。
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How to cite this protocol: Wei, Y. (2012). A Simple Preparation of RNA from Yeast by Hot Phenol for Northern Blot. Bio-protocol 2(12): e209. DOI: 10.21769/BioProtoc.209; Full Text



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