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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.

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RNA Extraction from RNase-Rich Senescing Leaf Samples

Plant Science > Plant molecular biology > RNA > RNA extraction
Author: 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, 4151 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, Senescence, Leaf, Arabidopsis

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



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