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RNA Extraction from RNase-Rich Senescing Leaf Samples
一种从富含RNA酶的衰老叶片样本中提取RNA的方法   

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Abstract

Isolation of intact, full-length high quality RNAs is essential for RNA sequencing, reverse transcription PCR analysis of gene expression as well as RNA gel blot analysis. This simple yet easy protocol is developed to meet this need; in addition to regular samples, this protocol is especially good for isolating RNAs from RNase-rich samples such as senescing leaves and ripening fruits (from which RNAs isolated using standard method are generally degraded to certain degree). The total RNA yield varies from 900 μg total RNA/g non-senescing leaves to 200 μg total RNA/g senescent leaves.

Keywords: RNA(RNA), Senescence(衰老), Leaf(叶), Arabidopsis(拟南芥)

Materials and Reagents

  1. Guanidinium thiocyanate (Thermo Fisher Scientific, catalog number:  BP221 )
  2. NaCitrate
  3. N-Lauroylsarcosine (Sigma-Aldrich, catalog number: L9150 )
  4. β-mercaptoethanol (β-ME)
  5. Glacial HAc
  6. NaOH
  7. Phenol (J.T.Baker®, catalog number:  2859 )
  8. Ethylenediaminetetraacetic acid (EDTA)
  9. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L5750 )
  10. Tris (hydroxymethyl) aminomethane
  11. Chloroform
  12. Isopropanol
  13. Diethylpyrocarbonate (DEPC)
  14. Ethanol
  15. Extraction buffer (EB) (see Recipes)
  16. 2 M NaAcetate (pH 4.0) (see Recipes)
  17. 2 M NaAcetate (pH 5.0) (DEPC treated) (see Recipes)
  18. H2O-saturated phenol (no buffer required) (see Recipes)
  19. Citrate-EDTA-SDS solution (CES) (see Recipes)
  20. Tris-EDTA-SDS solution (TES) (see Recipes)
  21. 100 ml 0.75 M NaCitrate (see Recipes)
  22. 60 ml 10% Sarkosyl (see Recipes)

Equipment

  1. Vortex Mixer  
  2. Beckman high speed centrifuge with JS13.1 rotor or the like
  3. 15-ml Corning centrifuge tube

Procedure

  1. 0.2-0.5 g sample tissue ground in liquid N2 (better grinding ensures a higher RNA yield).
  2. Add to 15-ml Corning centrifuge tube containing 5 ml EB (2.5 ml EB if sample is less than 0.2 g), vortex 2 min at room temperature.
  3. Add 0.5 ml 2 M NaAcetate (pH 4.0) to the tube, vortex 1 min at room temperature.
  4. Add 5 ml H2O-saturated phenol, vortex 1 min at room temperature.
  5. Add 1 ml chloroform, vortex 1 min at room temperature.
  6. Spin 10 min at 10,000 x g, 4 °C (8,000 rpm, JS13.1 rotor), transfer aqueous phase to a new 15-ml Corning centrifuge tube.
  7. Add equal vol. isopropanol to the new tube, screw cap and mix by inverting several times, keep the tube at -20 °C for >1 h.
  8. Spin 10 min at 3,000 x g, 4 °C (4,500 rpm JS13.1), discard supernatant.
  9. Add 2 ml CES or TES to redissolve pellet.
  10. Add 2 ml chloroform, vortex 1 min at room temperature.
  11. Spin 10 min at 3,000 x g, 4 °C, transfer aqueous to new tube.
  12. Add 1/10 vol. 2 M NaAcetate (pH 5.0), equal vol. isopropanol, pellet by spinning 10 min at 3,000 x g, 4 °C.
  13. Wash the pellet with 75% and 100% ethanol, respectively, air dry the pellet, and dissolve the pellet with 50-200 μl DEPC-treated CES or TES (for RT PCR or other analyses involving enzymes, DEPC-treated CE or TE without SDS should be used). The total RNA yield from senescent leaves is low, so a reduced final volume is suggested.

Recipes

  1. EB
    in 100 ml
    in 300 ml
    4 M Guanidinium thiocyanate (MW 118.2)
    47.28 g
    141.84 g
    25 mM NaCitrate (pH 7.0) (MW 294.10)
    3.33 ml of 0.75 M
    10 ml of 0.75 M
    0.5% Sarkosyl (N-Lauroylsarcosine, 293.4)
    5 ml of 10% Soln
    15 ml of 10%
    0.1 M β-ME (in 5 ml EB, add 35 μl conc)
    53.8 ml H2O

  2. 2 M NaAcetate (pH 4.0) (MW 82.03)
    11.49 ml glacial HAc (17.4 M)
    70 ml H2O
    Titrate w/ NaOH to pH 4.0 (this pH value is essential)
    ddH2O to 100 ml
  3. 2 M NaAcetate (pH 5.0) (MW 82.03) (DEPC treated)
    11.49 ml glacial HAc (17.4 M)
    70 ml H2O
    Titrate w/ NaOH to pH 5.0
    ddH2O to 100 ml
  4. H2O-saturated phenol (no buffer required)
  5. Citrate-EDTA-SDS solution(CES)
    for 100 ml
    10 mM NaCitrate(pH 7.0)
    1.333 ml 0.75 M NaCitrate (pH 7.0)
    (DEPC) 1 mM EDTA (pH 8.0)
    200 μl 0.5 M EDTA
    0.5% SDS
    2 ml 25% SDS
  6. Tris-EDTA-SDS solution (TES)
    10 mM Tris (pH 7.5)
    1 mM EDTA (pH 8.0)
    0.5% SDS   [DEPC]
  7. 100 ml 0.75 M NaCitrate
    15.761 g Citric acid (MW 210.14)
    80 ml ddH2O
    Titrate w/ NaOH to pH 7.0
    ddH2O to 100 ml
  8. 60 ml 10% Sarkosyl
    6 g N-lauroylsarcosine (MW 293.4)
    60 ml ddH2O
    65 °C stir

Acknowledgments

This protocol was initially developed from Chomczynski and Sacchi (1987) and Puissant and Houdebine (1990) at the Amasino Lab, University of Wisconsin-Madison and finalized at Cornell University. Su-Sheng Gan was a recipient of The Rockefeller Foundation Biotechnology Predoctoral Fellowship. The research was also partially supported by a Federal Formula Fund at Cornell University. The past and current members of the Gan Lab also greatly helped in improving the protocol.

References

  1. Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1): 156-159.
  2. Puissant, C. and Houdebine, L. M. (1990). An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Biotechniques 8(2): 148-149.

简介

Isolation of intact, full-length high quality RNAs is essential for RNA sequencing, reverse transcription PCR analysis of gene expression as well as RNA gel blot analysis. This simple yet easy protocol is developed to meet this need; in addition to regular samples, this protocol is especially good for isolating RNAs from RNase-rich samples such as senescing leaves and ripening fruits (from which RNAs isolated using standard method are generally degraded to certain degree). The total RNA yield varies from 900 μg total RNA/g non-senescing leaves to 200 μg total RNA/g senescent leaves.

关键字:RNA, 衰老, 叶, 拟南芥

材料和试剂

  1. 硫氰酸胍(Thermo Fisher Scientific,目录号:BP221)
  2. 柠檬酸钠
  3. N-月桂酰肌氨酸(Sigma-Aldrich,目录号:L9150)
  4. β-巯基乙醇(β-ME)
  5. 冰川HAc
  6. NaOH
  7. 苯酚(J.T.Baker ,目录号:2859)
  8. 乙二胺四乙酸(EDTA)
  9. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L5750)
  10. 三(羟甲基)氨基甲烷
  11. 氯仿
  12. 异丙醇
  13. 焦碳酸二乙酯(DEPC)
  14. 乙醇
  15. 提取缓冲液(EB)(参见配方)
  16. 2 M醋酸钠(pH 4.0)(参见配方)
  17. 2 M醋酸钠(pH 5.0)(DEPC处理)(参见配方)
  18. H 2 O饱和的苯酚(不需要缓冲液)(参见配方)
  19. 柠檬酸-EDTA-SDS溶液(CES)(参见配方)
  20. Tris-EDTA-SDS溶液(TES)(参见配方)
  21. 100ml 0.75M柠檬酸钠(见配方)
  22. 60 ml 10%Sarkosyl(参见配方)

设备

  1. 涡流混合器
  2. 带JS13.1转子的Beckman高速离心机等
  3. 15-ml Corning离心管

程序

  1. 在液体N 2中研磨0.2-0.5g样品组织(更好的研磨确保更高的RNA产率)。
  2. 加入到含有5ml EB(2.5ml EB,如果样品小于0.2g)的15-ml Corning离心管中,在室温下涡旋2分钟。
  3. 向管中加入0.5ml 2M乙酸钠(pH 4.0),在室温下涡旋1分钟。
  4. 加入5ml H 2 O饱和的苯酚,在室温下涡旋1分钟。
  5. 加入1ml氯仿,在室温下涡旋1分钟。
  6. 在10,000×g,4℃(8,000rpm,JS13.1转子)下旋转10分钟,将水相转移到新的15-ml Corning离心管中。
  7. 添加等体积。 异丙醇至新管,螺旋盖并通过颠倒数次混合,将管在-20℃下保持> 1小时。
  8. 在3,000×g,4℃(4,500rpm JS13.1)下旋转10分钟,弃去上清液。
  9. 加入2ml CES或TES以重新溶解沉淀。
  10. 加入2ml氯仿,在室温下涡旋1分钟。
  11. 在3,000xg,4℃下旋转10分钟,将水性转移到新管中。
  12. 加入1/10体积。 2 M醋酸钠(pH 5.0),等体积。 异丙醇,通过在3,000×g,4℃下旋转10分钟来沉淀。
  13. 分别用75%和100%乙醇洗涤沉淀,空气干燥沉淀,用50-200μlDEPC处理的CES或TES溶解沉淀(用于RT PCR或其他涉及酶,DEPC处理的CE或TE的分析 SDS应使用)。 衰老叶片的总RNA产量低,因此建议减少终体积。

食谱

  1. EB
    在100 ml
    在300ml
    4 M硫氰酸胍(MW 118.2)
    47.28克
    141.84克
    25mM柠檬酸钠(pH7.0)(MW294.10) 3.33ml 0.75M/dm 10ml的0.75M v/v
    0.5%肌氨酰(N-月桂酰肌氨酸,293.4) 5 ml 10%Soln
    15ml 10%
    0.1Mβ-ME(在5ml EB中,加入35μl浓) 53.8ml H 2 O v/v
  2. 2 M醋酸钠(pH 4.0)(MW 82.03)
    11.49 ml冰川HAc(17.4 M)
    70 ml H 2 O
    将pH值调整为pH 4.0 >(这个pH值是必需的)
    ddH 2 O至100 ml
  3. (pH5.0)(MW 82.03)(经DEPC处理) 11.49 ml冰川HAc(17.4 M)
    70 ml H 2 O
    将NaOH滴定至pH5.0
    ddH 2 O至100 ml
  4. H 2 O饱和的酚(不需要缓冲液)
  5. 柠檬酸-EDTA-SDS溶液(CES)
    为100毫升
    10mM柠檬酸钠(pH7.0) 1.333ml 0.75M柠檬酸钠(pH 7.0)
    (DEPC)1mM EDTA(pH8.0) 200μl0.5M EDTA
    0.5%SDS
    2ml 25%SDS
  6. Tris-EDTA-SDS溶液(TES)
    10mM Tris(pH7.5) 1mM EDTA(pH8.0) 0.5%SDS   [DEPC]
  7. 100ml 0.75M柠檬酸钠 15.761克柠檬酸(MW 210.14)
    80ml ddH 2 O 2 / 将w/NaOH滴定至pH7.0
    ddH 2 O至100ml
  8. 60 ml 10%Sarkosyl
    6克N-月桂酰肌氨酸(MW 293.4) 60ml ddH 2 O 2 65℃搅拌

致谢

该方案最初由Chomczynski和Sacchi(1987)和Puissant和Houdebine(1990)在Amasino Lab,Wisconsin-Madison大学开发,并在Cornell大学完成。 苏生甘获得洛克菲勒基金会生物技术前沿奖学金。 该研究也得到康奈尔大学联邦公式基金的部分支持。 甘氏实验室的过去和现任成员也大大有助于改进协议。

参考文献

  1. Chomczynski,P。和Sacchi,N。(1987)。 通过酸性硫氰酸胍 - 酚 - 氯仿萃取法分离RNA的单步法。 Anal Biochem 162(1):156-159。
  2. Puissant,C.and Houdebine,L.M。(1990)。 通过酸性硫氰酸胍 - 苯酚 - 氯仿萃取的单步分离方法的改进。 Biotechniques 8(2):148-149
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gan, S. (2012). RNA Extraction from RNase-Rich Senescing Leaf Samples. Bio-protocol 2(16): e248. DOI: 10.21769/BioProtoc.248.
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