Skip to main content
SpringerLink
Log in

Isolation of High-Quality RNA from Reaumuria soongorica, a Desert Plant Rich in Secondary Metabolites

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

Abbreviations

CTAB:

Cetyl trimethyl ammonium bromide

DEPC:

Diethyl pyrocarbonate

EDTA:

Ethylenediaminetetraacetic acid

LiCl:

Lithium chloride

NaAc:

Sodium acetate

PCI:

Phenol: chloroform: isoamylal alcohol

RT-PCR:

Reverse transcription polymerase chain reaction

PVPP:

Polyvinyl polypyrrolidone

SDS:

Sodium dodecyl sulfate

References

  1. Liu, W., Wang, B., Duan, C., & Li, B. (2005). A method for isolating functional RNA callus of Dendrobium candidum contented rich polysaccharides. Colloids and Surfaces B: Biointerfaces, 4, 259–262.

    Article  Google Scholar 

  2. Wang, X. C., Tain, W. M., & Li, Y. X. (2008). Development of an efficient protocol of RNA isolation from recalcitrant tree tissues. Molecular Biotechnology, 38, 57–64.

    Article  CAS  Google Scholar 

  3. Nap Bekesiovai, J. P., & Mlynarova, L. (1999). Plant Molecular Biology Reporter, 17, 269–277.

    Article  Google Scholar 

  4. Gehrig, H. H., Winter, K., Cushman, J., Borland, A., & Taybi, T. (2000). An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides. Plant Molecular Biology Reports, 18, 369–376.

    Article  CAS  Google Scholar 

  5. Geuna, F., Hartings, H., & Scienza, A. (1998). A new method for rapid extraction of high quality RNA from recalcitrant tissues of grapevines. Plant Molecular Biology Reporter, 16, 61–67.

    Article  CAS  Google Scholar 

  6. Wadsworth, G. J., Redinbaugh, M. G., & Scandalios, J. G. (1988). A procedure for the small scale isolation of plant RNA suitable for RNA blot analysis. Analytical Biochemistry, 172, 279–283.

    Article  CAS  Google Scholar 

  7. Vicient, C. M., & Delseny, M. (1999). Isolation of total RNA from Arabidopsis thaliana seeds. Analytical Biochemistry, 268, 412–413.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Li, B., Wang, B. C., Tang, K., Liang, Y. L., Wang, J., & Wei, J. (2006). A simple and convenient approach for isolating RNA from highly viscous plant tissue rich in polysaccharides. Colloids and Surfaces B: Biointerfaces, 49, 101–105.

    Article  CAS  Google Scholar 

  10. Kansal, R., Kuhar, K., Verma, I., Gupta, R. N., Gupta, V. K., & Koundal, K. R. (2008). Improved and convenient method of RNA isolation from polyphenols and polysaccharide rich plant tissues. Indian Journal of Experimental Biology, 46, 842–845.

    CAS  Google Scholar 

  11. Wang, L. M., & Stegemann, J. P. (2010). Extraction of high quality RNA from polysaccharide matrices using cetyltrimethylammonium bromide. Biomaterials, 31, 1612–1618.

    Article  CAS  Google Scholar 

  12. Loomis, W. D. (1974). Overcoming problems of phenolic and quinines in the isolation of plant enzymes and organelles. Methods in Enzymology, 31, 528–545.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Manickavelu, A., Kambara, K., Mishina, K., & Koba, T. (2007). An efficient method for purifying high quality RNA from wheat pistils. Colloids and Surfaces B: Biointerfaces, 54, 254–258.

    Article  CAS  Google Scholar 

  15. Liu, J. Q., Qiu, M. X., Pu, J. C., & Lu, Z. M. (1982). The typical extreme xerophyte Reaumuria soongorica in the desert of China. Acta Botanica Sinica, 24, 485–488.

    Google Scholar 

  16. Liu, Y. B., Zhang, T. G., An, L. Z., & Wang, G. (2006). Isolation of RNA using modified method of CTAB from dehydrated and nondehydrated Reaumuria soongorica tissues. Journal of Desert Research, 26, 600–603.

    Google Scholar 

  17. Liu, Y. B., Zhao, X., & Tan, H. J. (2008). Induction and proliferation of callus in desert plant Reaumuria soongorica. Journal of Desert Research, 28, 254–258.

    Google Scholar 

  18. Asif, M. H., Dhawan, P., & Nath, P. (2000). A simple procedure for the isolation of high quality RNA from ripening banana fruit. Plant Molecular Biology Reporter, 18, 109–115.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Manning, K. (1990). Isolation of nucleic acids from plants by differential solvent precipitation. Analytical Biochemistry, 195, 45–50.

    Article  Google Scholar 

  21. Li, R., Mock, R., Huang, Q., Abad, J., Hartung, J., & Kinard, G. (2008). A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. Journal of Virological Methods, 154, 48–55.

    Article  CAS  Google Scholar 

  22. Azevedo, H., Lino-Neto, T., & Tavares, R. M. (2003). An improved method for high-quality RNA isolation from needles of adult maritime pine trees. Plant Molecular Biology Reporter, 21, 333–338.

    Article  CAS  Google Scholar 

  23. Jaakola, L., Pirttila, A. M., Halonen, M., & Hohtola, A. (2001). Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit. Molecular Biotechnology, 19, 201–203.

    Article  CAS  Google Scholar 

  24. Porebski, S., Bailey, L., & Baum, B. (1997). Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter, 15, 8–15.

    Article  CAS  Google Scholar 

  25. Chang, S., Puryear, J., & Cairney, J. (1993). A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter, 11, 113–116.

    Article  CAS  Google Scholar 

  26. Fang, G., Hammar, S., & Grumet, R. (1992). A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biofeedback, 13, 52–54.

    CAS  Google Scholar 

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

    Article  Google Scholar 

  28. Salzman, R. A., Fujita, T., Salzman, K. Z., Hasegawa, 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 

  29. Venkatachalam, P., Thanseem, I., & Thulaseedharan, A. (1999). A rapid and efficient method for isolation of RNA from bark tissues of Hevea brasiliensis. Current Science, 77, 635–637.

    CAS  Google Scholar 

  30. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual (2nd ed.). New York: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  31. Gareth, P., Asuncion, L. L., Marta, V., & Ester, S. (2006). Simple and rapid RNA extraction from freeze-dried tissue of brown algae and seagrasses. European Journal of Phycology, 41, 97–104.

    Article  Google Scholar 

  32. Birnboim, H. C. (1992). Extraction of high molecular weight RNA and DNA from cultured mammalian cells. Methods Enzymology, 216, 154–160.

    Article  CAS  Google Scholar 

  33. Chen, G. Y. J., Jin, S., & Goodwin, P. H. (2000). An improved method for the isolation of total RNA from Malva pusilla tissues infected with Colletotrichum gloeosporioides. Journal of Phytopathology, 148, 57–60.

    CAS  Google Scholar 

  34. Dellacorte, C. (1994). Isolation of nucleic acids from the sea anemone Condylactis gigantea (Cnidaria: anthozoa). Tissue and Cell, 26, 613–619.

    Article  CAS  Google Scholar 

  35. Mannan, M. A., Sharma, S., & Ganesan, K. (2009). Total RNA isolation from recalcitrant yeast cells. Analytical Biochemistry, 389, 77–79.

    Article  Google Scholar 

Download references

Acknowledgments

This study was supported by the National Key Technologies R&D Program of the 11th five-year plan (2007BAD46B) and the National Natural Science Foundation of China (Grant no. 40901019/D010102).

Author information

Authors and Affiliations

  1. Key Laboratory of Ecohydrology and of Inland River Basin, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000, People’s Republic of China

    Xiaohua Wang, Honglang Xiao & Fang Wang

  2. Laboratory of Plant Ecophysiology and Biotechnology, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000, People’s Republic of China

    Guoxiong Chen, Xin Zhao, Chenghong Huang & Cuiyun Chen

Authors
  1. Xiaohua Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Honglang Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guoxiong Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xin Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chenghong Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Cuiyun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Honglang Xiao.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, X., Xiao, H., Chen, G. et al. Isolation of High-Quality RNA from Reaumuria soongorica, a Desert Plant Rich in Secondary Metabolites. Mol Biotechnol 48, 165–172 (2011). https://doi.org/10.1007/s12033-010-9357-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-010-9357-3

Keywords

Search

Navigation