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High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith

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Abstract

Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of β-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 μg total RNA per gram of cells was obtained with an average purity measured as A260/A280 of 2.09. A titer of 105 cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.

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References

  1. Gill, C. O., Hall, M. J., & Ratledge, C. (1977). Lipid accumulation in an oleaginous yeast (Candida 107) growing on glucose in single-stage continuous culture. Applied and Environmental Microbiology, 33, 231–239.

    CAS  Google Scholar 

  2. Meng, X., Yang, J. M., Xu, X., Zhang, L., Nie, Q. J., & Xian, M. (2009). Biodiesel production from oleaginous microorganisms. Renewable Energy, 34, 1–5.

    Article  Google Scholar 

  3. Vicente, G., Bautista, L. F., Rodriguez, R., Gutierrez, F. J., Sadaba, I., Ruiz-Vazquez, R. M., et al. (2009). Biodiesel production from biomass of an oleaginous fungus. Biochemical Engineering Journal, 48, 22–27.

    Article  CAS  Google Scholar 

  4. Ratledge, C. (2002). Regulation of lipid accumulation in oleaginous micro-organisms. Biochemical Society Transactions, 30, 1047–1050.

    Article  CAS  Google Scholar 

  5. Ratledge, C., & Wynn, J. P. (2002). The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Advances in Applied Microbiology, 51, 1–51.

    Article  CAS  Google Scholar 

  6. Liu, B., Sun, Y., Li, Y. H., & Zhao, Z. B. (2005). Progress on microbial glyceride biosynthesis and metabolic regulation in oleaginous microorganisms. Acta Microbiologica Sinica, 45, 153–156.

    CAS  Google Scholar 

  7. Tang, W., Zhang, S. F., Wang, Q., Tan, H. D., & Zhao, Z. K. (2009). The isocitrate dehydrogenase gene of oleaginous yeast Lipomyces starkeyi is linked to lipid accumulation. Canadian Journal of Microbiology, 55, 1062–1069.

    Article  CAS  Google Scholar 

  8. Turcotte, G., & Kosaric, N. (1988). Biosynthesis of lipids by Rhodosporidium toruloides ATCC 10788. Journal of Biotechnology, 8, 211–237.

    Article  Google Scholar 

  9. Li, Y. H., Zhao, Z. B., & Bai, F. W. (2007). High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme and Microbial Technology, 41, 312–317.

    Article  Google Scholar 

  10. Liu, H. W., Zhao, X., Wang, F. J., Li, Y. H., Jiang, X. N., Ye, M. L., et al. (2009). Comparative proteomic analysis of Rhodosporidium toruloides during lipid accumulation. Yeast, 26, 553–566.

    Article  Google Scholar 

  11. Wang, X. X., Wang, B., Liu, L. J., Cui, X. P., Yang, J. Y., Wang, H., et al. (2010). Isolation of high quality RNA and construction of a suppression subtractive hybridization library from ramie (Boehmeria nivea L. Gaud.). Molecular Biology Reports, 37, 2099–2103.

    Article  CAS  Google Scholar 

  12. Marguerat, S., & Bahler, J. (2010). RNA-seq: From technology to biology. Cellular and Molecular Life Sciences, 67, 569–579.

    Article  CAS  Google Scholar 

  13. Baker, S. S., Rugh, C. L., & Kamalay, J. C. (1990). RNA and DNA isolation from recalcitrant plant tissues. Biotechniques, 9, 268–272.

    CAS  Google Scholar 

  14. 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 

  15. Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  16. Portillo, M., Fenoll, C., & Escobar, C. (2006). Evaluation of different RNA extraction methods for small quantities of plant tissue: Combined effects of reagent type and homogenization procedure on RNA quality-integrity and yield. Physiologia Plantarum, 128, 1–7.

    Article  CAS  Google Scholar 

  17. Wang, X. C., Tian, 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 

  18. Zhong, Z. P., & Wu, N. H. (1997). Stability of mRNA in eukaryote. Progress in Biotechnology, 17, 34–38.

    Google Scholar 

  19. Murao, S., Yamamoto, R., & Arai, M. (1976). Isolation and identification of red yeast cell wall lytic enzyme producing microorganism. Agricultural and Biological Chemistry, 40, 23–26.

    CAS  Google Scholar 

  20. de Coana, Y. P., Parody, N., Fernandez-Caldas, E., & Alonso, C. A. (2010). Modified protocol for RNA isolation from high polysaccharide containing Cupressus arizonica Pollen. Applications for RT-PCR and phage display library construction. Molecular Biotechnology, 44, 127–132.

    Article  CAS  Google Scholar 

  21. Ghangal, R., Raghuvanshi, S., & Sharma, P. C. (2009). Isolation of good quality RNA from a medicinal plant seabuckthorn, rich in secondary metabolites. Plant Physiology and Biochemistry, 47, 1113–1115.

    Article  CAS  Google Scholar 

  22. 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 

  23. Schmitt, M. E., Brown, T. A., & Trumpower, B. L. (1990). A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Research, 18, 3091–3092.

    Article  CAS  Google Scholar 

  24. Li, Y., Wang, W. Y., Du, X. L., & Yuan, Q. P. (2010). An improved RNA isolation method for filamentous fungus Blakeslea trispora rich in polysaccharides. Applied Biochemistry and Biotechnology, 160, 322–327.

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  26. Wang, K., Wang, Y., Bao, Y. L., Meng, X. Y., Wu, Y., & Li, Y. X. (2006). A method for high-quality RNA extraction from Panax ginseng plant tissues. Progress in Biochemistry and Biophysics, 33, 95–99.

    Google Scholar 

  27. Hilz, H., Wiegers, U., & Adamietz, P. (1975). Stimulation of proteinase K action by denaturing agents: Application to the isolation of nucleic acids and the degradation of ‘masked’ proteins. European Journal of Biochemistry, 56, 103–108.

    Article  CAS  Google Scholar 

  28. Chan, C. X., Teo, S. S., Ho, C. L., Othman, R. Y., & Phang, S. M. (2004). Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta). Journal of Applied Phycology, 16, 297–301.

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

  30. Thanh, T., Omar, H., Abdullah, M. P., Chi, V. T. Q., Noroozi, M., Ky, H., et al. (2009). Rapid and effective method of RNA isolation from green microalga Ankistrodesmus convolutus. Molecular Biotechnology, 43, 148–153.

    Article  CAS  Google Scholar 

  31. Wang, X. W., Xiong, A. S., Yao, Q. H., Zhang, Z., & Qiao, Y. S. (2010). Direct isolation of high-quality low molecular weight RNA of pear peel from the extraction mixture containing nucleic acid. Molecular Biotechnology, 44, 61–65.

    Article  CAS  Google Scholar 

  32. 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 

  33. Sharma, A. D., Gill, P. K., & Singh, P. (2003). RNA isolation from plant tissues rich in polysaccharides. Analytical Biochemistry, 314, 319–321.

    Article  CAS  Google Scholar 

  34. Singh, G., Kumar, S., & Singh, P. (2003). A quick method to isolate RNA from wheat and other carbohydrate-rich seeds. Plant Molecular Biology Reporter, 21, 93a–93f.

    Article  Google Scholar 

  35. Yao, J. T., Fu, W. D., Wang, X. L., & Duan, D. L. (2009). Improved RNA isolation from Laminaria japonica Aresch (Laminariaceae, Phaeophyta). Journal of Applied Phycology, 21, 233–238.

    Article  CAS  Google Scholar 

  36. de Jong, M., Rauwerda, H., Bruning, O., Verkooijen, J., Spaink, H. P., & Breit, T. M. (2010). RNA isolation method for single embryo transcriptome analysis in zebrafish. BMC Research Notes, 3, 73–78.

    Article  Google Scholar 

  37. Kocabiyik, S. (1996). Modified method for RNA isolation from bacteria by guanidinium thiocyanate-cesium chloride centrifugation. Biotechniques, 18, 570–572.

    Google Scholar 

  38. Wu, Y., Llewellyn, D. J., & Dennis, E. S. (2002). A quick and easy method for isolating good-quality RNA from cotton (Gossypium hirsutum L.) tissues. Plant Molecular Biology Reporter, 20, 213–218.

    Article  CAS  Google Scholar 

  39. Wang, D. H., Wang, B. C., Li, B., Duan, C. R., & Zhang, J. (2004). Extraction of total RNA from Chrysanthemum containing high levels of phenolic and carbohydrates. Colloids and Surfaces B-Biointerfaces, 36, 111–114.

    Article  Google Scholar 

  40. Pearson, G., Lago-Leston, A., Valente, M., & Serrao, E. (2006). Simple and rapid RNA extraction from freeze-dried tissue of brown algae and seagrasses. European Journal of Phycology, 41, 97–104.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (No. K2006A22).

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Authors and Affiliations

  1. Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, 116023, People’s Republic of China

    Fan Yang, Haidong Tan, Yongjin Zhou, Xinping Lin & Sufang Zhang

  2. Graduate University of the Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Fan Yang, Yongjin Zhou & Xinping Lin

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  1. Fan Yang
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  2. Haidong Tan
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Correspondence to Sufang Zhang.

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Yang, F., Tan, H., Zhou, Y. et al. High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith. Mol Biotechnol 47, 144–151 (2011). https://doi.org/10.1007/s12033-010-9322-1

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