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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

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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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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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