Molecular Biotechnology

, Volume 38, Issue 1, pp 57–64

Development of an Efficient Protocol of RNA Isolation from Recalcitrant Tree Tissues

Original Paper

Abstract

Isolation of RNA from recalcitrant tree tissues has been problematic due to large amounts of secondary metabolites and interfering compounds in their cells. We have developed an efficient RNA extraction method, which yielded high-quality RNA preparations from tissues of the lychee tree. The method reported here utilized EDTA, LSS, and CTAB to successfully inhibit RNase activities. It was found that a high ionic strength brought about by 2 M NaCl was necessary. In addition, secondary metabolites and other interfering compounds were effectively removed using sodium borate and PVPP under a deoxidized condition. The quality of purified RNA was tested by both RACE and Northern blotting analysis, ensuring that the RNA could be used for subsequent gene expression analysis. This method has been successfully applied to purify RNA from 15 other plant species. In conclusion, the protocol reported here is expected to have excellent applications for RNA isolation from recalcitrant plant tissues.

Keywords

Lychee Recalcitrant plant tissues RNA extraction method Trees 

Abbreviations

CMO

Choline monooxygenase

CTAB

Cetyltrimethylammonium bromide

DEPC

Diethypyrocarbonate

EDTA

Ethylenediaminetetraacetic acid

LSS

N-lauroyl sarcosine sodium

PVPP

Polyvinylpolypyrrolidone

RACE

Rapid amplification of cDNA ends

References

  1. 1.
    Ainsworth, C. (1994). Isolation of RNA from floral tissue of Rumex acetosa (Sorrel). Plant Molecular Biology Reporter, 12, 198–203.Google Scholar
  2. 2.
    Birtic, S., & Kranner, I. (2006). Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds. Phytochemical Analysis, 17, 144–148.PubMedCrossRefGoogle Scholar
  3. 3.
    Chang, S., Puryear, J., & Caimey, J. (1993). A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter, 11, 113–116.Google Scholar
  4. 4.
    Chomczynski, P., & Sacchi, N. (1987). Single step method of RNA isolation by acid guanidinium thiocyanale-phenol-chloroform extraction. Analytical Biochemistry, 162, 156–159.PubMedCrossRefGoogle Scholar
  5. 5.
    Chomczynski, P. (1993). A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques, 15, 532–537.PubMedGoogle Scholar
  6. 6.
    Claros, M. G., & Canovas, F. M. (1998). Rapid high quality RNA preparation from pine seedlings. Plant Molecular Biology Reporter, 16, 9–18.CrossRefGoogle Scholar
  7. 7.
    Davy, A. J., Bishop, G. F., & Costa, C. B. (2001). Salicornia L. (Salicornia pusilla J. Woods, S. ramosissima J. Woods, S. europaea L., S. obscura P.W. Ball & Tutin, S. nitens P.W. Ball & Tutin, S. fragilis P.W. Ball & Tutin and S. dolichostachya Moss). Journal of Ecology, 89, 681–707.CrossRefGoogle Scholar
  8. 8.
    Geuna, F., Hartings, H., & Scienza, A. (1998). A new method for rapid extraction of high quality RNA from recalcitrant tissues of grapevine. Plant Molecular Biology Reporter, 16, 61–67.CrossRefGoogle Scholar
  9. 9.
    Ignacio, I. F., Leticia, P. E., Blondy, C. C., & Monica, R. C. (2006). Extraction of high-quality, melanin-free RNA from Mycosphaerella fijiensis for cDNA preparation. Molecular Biotechnology, 34, 45–50.CrossRefGoogle Scholar
  10. 10.
    Ildiko, B., Janpeter, N., & Mlynarova, L. (1999). Isolation of high quality DNA and RNA from leaves of the carnivorous plant Drosera rotundifolia. Plant Molecular Biology Reporter, 17, 269–277.CrossRefGoogle Scholar
  11. 11.
    Jelle, D. K., Isabel, R. R., Erik, V. B., Arne, H., & Denis, D. K. (2006). Efficient extraction of High-Quality total RNA from various hop tissues. Preparative Biochemistry and Biotechnology, 36, 355–362.CrossRefGoogle Scholar
  12. 12.
    John, M. E. (1992). An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Research, 20, 2381.PubMedCrossRefGoogle Scholar
  13. 13.
    Kiefer, E., Werener, H., & Dieter, E. (2000). A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites. Plant Molecular Biology Reporter, 18, 33–39.Google Scholar
  14. 14.
    Kim, S. H., & Hamada, T. (2005). Rapid and reliable method of extracting DNA and RNA from sweetpotato, Ipomoea batatas (L). Lam. Biotechnology Letter, 27, 1841–1845.CrossRefGoogle Scholar
  15. 15.
    Li, J. H., Tang, G. H., Song, C. Y., Chen, M. J., Feng, Z. Y., & Pan, Y. J. (2006). A simple, rapid and effective method for total RNA extraction from Lentinula edodes. Biotechnology Letters, 28, 1193–1197.PubMedCrossRefGoogle Scholar
  16. 16.
    Liu, J. J., Goh, C. J., Loh, C. S., Liu, P., & Pua, E. C. (1998). A method for isolation of total RNA from fruit tissues of banana. Plant Molecular Biology Reporter, 16, 1–6.Google Scholar
  17. 17.
    Logemann, J., Schell, J., & Willmitzer, L. (1987). Improved method for isolation of RNA from plant tissue. Analytical Biochemistry, 163, 16–20.PubMedCrossRefGoogle Scholar
  18. 18.
    Malnoy, M., Reynoird, J. P., Mourgues, F., Cheverau, E., & Simoneau, P. (2001). A method for isolation total RNA from pear. Plant Molecular Biology Reporter, 19, 69–74.CrossRefGoogle Scholar
  19. 19.
    Manickavelu, A., Kambara, K., Mishina, K., & Koba, T. (2007). An efficient method for purifying high quality RNA from wheat pistils. Colloid Surface, B54, 254–258.Google Scholar
  20. 20.
    Meisel, L., Fonseca, B., Gonzalez, S., Baezayates, R., Cambiazo, V., Campos, R., Gonzalez, M., Orelana, A., Retamales, J., & Silva, H. (2005). A rapid and efficient method for purifying high quality total RNA from peaches (prunus persica) for functional genomics analyses. Biological Research, 38, 83–88.PubMedCrossRefGoogle Scholar
  21. 21.
    Portillo, M., Fenoll, C., & Escobar, C. (2006). Evaluation of different RNA extraction methods for small quantities of plant tissue. Physiologia Plantarum, 128, 1–7.CrossRefGoogle Scholar
  22. 22.
    Riehl, T. E., & Ungar, I. A. (1982). Growth and ion accumulation in Salicornia europaea under saline field conditions. Oecologia, 54, 193–199.CrossRefGoogle Scholar
  23. 23.
    Russell, B., Rathinasabapathi, B., & Hanson, A. D. (1998). Osmotic stress induces expression of choline monooxygenase in sugar beet and amaranth. Plant Physiology, 116, 859–865.PubMedCrossRefGoogle Scholar
  24. 24.
    Salzman, R. A., Fujiya, T. K., Salzman, Z., Hasgawa, P. M., & Bressan, R. A. (1999). An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Molecular Biology Reporter, 17, 11–17.CrossRefGoogle Scholar
  25. 25.
    Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (pp. 343–388). Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press.Google Scholar
  26. 26.
    Shi, H. Z., & Bressan, R. (2006). RNA extraction. Methods in Molecular Biology, 323, 345–348.PubMedGoogle Scholar
  27. 27.
    Su, X., & Gibor, A. (1988). A method for RNA isolation from marine macro-algae. Analytical Biochemistry, 174, 650–657.PubMedCrossRefGoogle Scholar
  28. 28.
    Wan, C. Y., & Wilkins, T. A. (1994). A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Analytical Biochemistry, 223, 7–12.PubMedCrossRefGoogle Scholar
  29. 29.
    Xu, Q., Wen, X. P., Tao, N. G., Hu, Z. Y., Yue, H. L., & Deng, X. X. (2006). Extraction of high quality of RNA and construction of a suppression subtractive hybridization (SSH) library from chestnut rose (Rosa roxburghii Tratt). Biotechnology Letters, 28, 587–591.PubMedCrossRefGoogle Scholar
  30. 30.
    Yamashita, Y., Sakurai, T., Kuno, N., Uchida, K., & Yokobayshi, T. (2005). RNA extraction method, RNA extraction reagent, and method for analyzing biological materials. United States Patent, 981521.Google Scholar
  31. 31.
    Zeng, Y., & Yang, T. (2002). RNA isolation from highly viscous samples rich in polyphenols and polysaccharides. Plant Molecular Biology Reporter, 20, 415–417.Google Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  1. 1.Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of BotanyChinese Academy of SciencesBeijingP.R. China
  2. 2.Key Laboratory of Ministry of Agriculture for Tropical Crops PhysiologyChinese Academy of Tropical Agricultural SciencesDanzhouP.R. China

Personalised recommendations