欢迎您, 登录 | 注册

首页 | English

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

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

植物科学 > 植物分子生物学 > RNA > RNA 提取
作者: Susheng Gan
Susheng GanAffiliation: Horticulture Section and Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, USA
For correspondence: sg288@cornell.edu
Bio-protocol author page: a83
Vol 2, Iss 16, 8/20/2012, 4168 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.248

[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.


How to cite this protocol: Gan, S. (2012). RNA Extraction from RNase-Rich Senescing Leaf Samples. Bio-protocol 2(16): e248. DOI: 10.21769/BioProtoc.248; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook