Molecular Biotechnology

, Volume 38, Issue 1, pp 57–64

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

Original Paper

DOI: 10.1007/s12033-007-0073-6

Cite this article as:
Wang, X., Tian, W. & Li, Y. Mol Biotechnol (2008) 38: 57. doi:10.1007/s12033-007-0073-6

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.

Keywords

LycheeRecalcitrant plant tissuesRNA extraction methodTrees

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

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  1. 1.Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of BotanyChinese Academy of SciencesBeijingP.R. China
  2. 2.Key Laboratory of Ministry of Agriculture for Tropical Crops PhysiologyChinese Academy of Tropical Agricultural SciencesDanzhouP.R. China