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

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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.

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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. Ainsworth, C. (1994). Isolation of RNA from floral tissue of Rumex acetosa (Sorrel). Plant Molecular Biology Reporter, 12, 198–203.

    CAS  Google Scholar 

  2. Birtic, S., & Kranner, I. (2006). Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds. Phytochemical Analysis, 17, 144–148.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  4. Chomczynski, P., & Sacchi, N. (1987). Single step method of RNA isolation by acid guanidinium thiocyanale-phenol-chloroform extraction. Analytical Biochemistry, 162, 156–159.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  6. Claros, M. G., & Canovas, F. M. (1998). Rapid high quality RNA preparation from pine seedlings. Plant Molecular Biology Reporter, 16, 9–18.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  12. John, M. E. (1992). An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Research, 20, 2381.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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. Logemann, J., Schell, J., & Willmitzer, L. (1987). Improved method for isolation of RNA from plant tissue. Analytical Biochemistry, 163, 16–20.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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. 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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  22. Riehl, T. E., & Ungar, I. A. (1982). Growth and ion accumulation in Salicornia europaea under saline field conditions. Oecologia, 54, 193–199.

    Article  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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. Shi, H. Z., & Bressan, R. (2006). RNA extraction. Methods in Molecular Biology, 323, 345–348.

    PubMed  CAS  Google Scholar 

  27. Su, X., & Gibor, A. (1988). A method for RNA isolation from marine macro-algae. Analytical Biochemistry, 174, 650–657.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

  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 

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Acknowledgments

This research was supported by the National High Technology and Research Development Program of China (“863” project) (Grant No. 2007AA091705) and the Key Directional Research Project of CAS (Grant No. KSCX2-YW-N-003 and 013). We thank Drs Hongjie Li and Xuejun Hua for their critical reading of this paper. We thank in particular Professor Bo Liu at University of California-Davis for his major revisions and language polish of the whole article.

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Authors and Affiliations

  1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, P.R. China

    Xuchu Wang & Yinxin Li

  2. Key Laboratory of Ministry of Agriculture for Tropical Crops Physiology, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan, P.R. China

    Xuchu Wang & Weimin Tian

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  1. Xuchu Wang
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  2. Weimin Tian
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Correspondence to Yinxin Li.

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Wang, X., Tian, W. & Li, Y. Development of an Efficient Protocol of RNA Isolation from Recalcitrant Tree Tissues. Mol Biotechnol 38, 57–64 (2008). https://doi.org/10.1007/s12033-007-0073-6

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  • DOI: https://doi.org/10.1007/s12033-007-0073-6

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