Molecular Biotechnology

, Volume 37, Issue 3, pp 220–224 | Cite as

Isolation and Purification of RNA from Tissues Rich in Polyphenols, Polysaccharides, and Pigments of Annatto (Bixa orellana L.)

  • Simone M. Rodrigues
  • Virgínia L. F. Soares
  • Tahise M. de Oliveira
  • Abelmon S. Gesteira
  • Wagner C. Otoni
  • Marcio G. C. CostaEmail author
Original Paper


The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library.


RNA extraction MATAB (mixed alkyl trimethyl ammonium bromide) RT-PCR cDNA library 



This research was supported by CNPq (Research grant no. 473619/04-04 to WCO) and BNB (Research grant no. 2004-1-22 to MGCC). We gratefully acknowledge the Ph.D. scholarships to S. M. Rodrigues and V. L. F. Soares by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), respectively. We thank F. T. S. Nogueira (Cold Spring Harbor Laboratory, USA) for the critical reading of the manuscript. T. M. de Oliveira is recipient of a PIBIC fellowship from CNPq.


  1. 1.
    Lawrence, B. M., & Hogg, J. W. (1973). Ishwarane in Bixa orellana leaf oil. Phytochemistry, 12, 2995.CrossRefGoogle Scholar
  2. 2.
    Harbone, J. B. (1975). Flavonoid bisulfates and their co-occurrences with ellagic acid in the Bixaceae, Frankeniaceae, and related families. Phytochemistry, 14, 1331–1337.CrossRefGoogle Scholar
  3. 3.
    Jondiko, I. J. O., & Pattenden, G. (1989). Terpenoids and an apocarotenoid from seeds of Bixa orellana. Phytochemistry, 28, 3159–3162.CrossRefGoogle Scholar
  4. 4.
    Frega, N., Mozzon, M., & Bocci, F. (1998). Identification and estimation of tocotrienols in the annatto lipid fraction by gas chromatography mass spectrometry. Journal of the American Oil Chemists’ Society, 75, 1723–1727.CrossRefGoogle Scholar
  5. 5.
    Pino, J. A., & Correa, M. T. (2003). Chemical composition of the essential oil from annatto (Bixa orellana L.) seeds. Journal of Essential Oil Research, 15, 66–67.Google Scholar
  6. 6.
    Satyanarayana, A., Rao, P. G. P., & Rao, D. G. (2003). Chemistry, processing and toxicology of annatto (Bixa orellana L.). Journal of Food and Science Technology, 40, 131–141.Google Scholar
  7. 7.
    Shilpi, J. A., Taufiq-Ur-Rahman, Md., Uddin, S. J., Alam, Md. S., Sadhu, S. K., & Seidel, V. (2006). Preliminary pharmacological screening of Bixa orellana L. leaves. Journal of Ethnopharmacology, 108, 264–271.PubMedCrossRefGoogle Scholar
  8. 8.
    Giuliano, G., Rosati, C., & Bramley, P. M. (2003). To dye or not to dye: Biochemistry of annatto unveiled. Trends in Biotechnology, 21, 513–516.PubMedCrossRefGoogle Scholar
  9. 9.
    Mercadante, A. Z., & Pfander, H. (1998). Carotenoids from annatto: A review. Recent Research in Developmental and Agricultural Food Chemistry, 2, 79–91.Google Scholar
  10. 10.
    Bouvier, F., Dogbo, O., & Camara, B. (2003). Biosynthesis of the food and cosmetic plant pigment bixin (annatto). Science, 300, 2089–2091.PubMedCrossRefGoogle Scholar
  11. 11.
    Echevarria-Machado, I., Sanchez-Cach, L. A., Hernandez-Zepeda, C., Rivera-Madrid, R., & Moreno-Valenzuela, O. A. (2005). A simple and efficient method for isolation of DNA in high mucilaginous plant tissues. Molecular Biotechnology, 31, 129–135.PubMedCrossRefGoogle Scholar
  12. 12.
    Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry, 162, 156–159.PubMedCrossRefGoogle Scholar
  13. 13.
    De Vries, S., Hoge, H., & Biseeing, T. (1988). Isolation of total and polysomal RNA from plant tissues. Plant Molecular Biology Manual, B6, 64–71.Google Scholar
  14. 14.
    Hu, C. G., Honda, C., Kita, M., Zhang, Z. L., Tsuda, T., & Moriguchi, T. (2002). A simple protocol for RNA isolation from fruit trees containing high levels of polysaccharides and polyphenol compounds. Plant Molecular Biology Reporter, 20, 69a–69g.Google Scholar
  15. 15.
    Tao, N., Cheng, Y., Xu, J., Xu, Q., & Deng, X. (2004). An effective protocol for the isolation of RNA from the pulp of ripening citrus fruits. Plant Molecular Biology Reporter, 22, 305a–305f.CrossRefGoogle Scholar
  16. 16.
    Narváez, J. A., Flores-Peres, P., Herrera-Valencia, V., Castillo, F., Ku-Cauich, R., Canto-Canché, B. B., Buzzy, N. S., & Rivera-Madrid, R. (2001). Development of molecular techniques for studying the metabolism of carotenoids in Bixa orellana L. HortScience, 36, 982–986.Google Scholar
  17. 17.
    Gesteira, A. S., Micheli, F., Ferreira, C. F., & Cascardo, J. C. M. (2003) Isolation and purification of functional total RNA from different organs of cacao tree during its interaction with the pathogen Crinipellis perniciosa. BioTechniques, 35, 494–500.Google Scholar
  18. 18.
    Schultz, D. J., Craig, R., Cox-Foster, D. L., Mumma, R. O., & Medford, J. I. (1994). RNA isolation from recalcitrant plant tissue. Plant Molecular Biology Reporter, 12, 310–316.Google Scholar
  19. 19.
    Burgos, R. C., Chiang, V. L., Zhang, X. H., Campbell, E. R., Podila, G. K., & Campbell, W. H. (1995). RNA isolation from plant tissues recalcitrant to extraction in guanidine. BioTechniques, 19, 734–737.Google Scholar
  20. 20.
    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.Google Scholar
  21. 21.
    Chi-Manzanero, B., Robert, M. L., & Rivera-Madrid, R. (2000). Extraction of total RNA from a high pigment content plant marigold (Tagetes erecta). Molecular Biotechnology, 16, 17–21.PubMedCrossRefGoogle Scholar
  22. 22.
    Wang, S. X., Hunter, W., & Plant, H. (2000). Isolation and purification of functional total RNA from woody branches and needles of Sitka and White Spruce. BioTechniques, 28, 292–296.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Simone M. Rodrigues
    • 1
  • Virgínia L. F. Soares
    • 2
  • Tahise M. de Oliveira
    • 2
  • Abelmon S. Gesteira
    • 2
  • Wagner C. Otoni
    • 1
  • Marcio G. C. Costa
    • 2
    Email author
  1. 1.Plant Biology DepartmentFederal University of ViçosaVicosaBrazil
  2. 2.Biological Sciences DepartmentState University of Santa CruzIlheusBrazil

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