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An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis
Published on: January 11, 2017
A reliable and efficient method for total rna isolation from various members of spurge family (Euphorbiaceae)
Jia Xu1, Mahender Aileni, Sadanandam Abbagani
1National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.
Introduction:
It is prerequisite and crucial to extract RNA with high quality and integrity in order to carry out molecular biology studies in any plant species of a family. Euphorbiaceae members are known for high levels of their waxes, oils with polysaccharides, polyphenolics and secondary metabolites. These conditions are recognised to interfere unfavourably with various methodologies of RNA isolation.
Objective:
To develop a simple, rapid and reproducible cetyltrimethylamonium bromide (CTAB)-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from various recalcitrant Euphorbiaceae member plant tissues such as from tree leaves (Hevea brasilensis), woody shrubs leaves (Ricinus communis, Jatropha curcas, Manihot esculenta) and storage root tissue (M. esculenta).
Methodology:
Simple modifications and fast steps were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C at 12000 rpm for 10 min, the sample weight was decreased and usage of spermidine and LiCl was omitted, reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with various RNA isolation methods intended for use with plants rich in polysaccharides and secondary metabolites.
Results:
The procedure can be completed within 2 h and many samples can be processed at the same time. RNA of high quality could be isolated from all the tissues of species that we tried. The isolated RNA from different species served as a robust template for RT-PCR analysis.
Conclusion:
The study has shown that the improvement of a CTAB-based protocol allows the rapid isolation of high-quality RNA from various recalcitrant Euphorbiaceae members.
