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Comparison of Methods for Isolating High-Quality RNA from Leaves of Grapevine

¹ Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557;

^* Corresponding author [Email: cramer{at}unr.edu; fax 775-784-1650]

High concentrations of polyphenols and polysaccharides make it challenging to extract high-quality RNA from grape organs. To determine an optimal protocol for grape leaves, 15 different methods of RNA extraction were evaluated based on cost, time to complete the extraction, and quality of the RNA isolated. The addition of specific compounds to the extraction buffer to remove polyphenols and polysaccharides is often critical for downstream applications such as polymerase chain reaction (PCR) and microarray hybridization. RNA quality was assessed using spectrophotometric methods, formaldehyde-agarose gel electrophoresis, reverse transcription (RT)-PCR reactions, and Agilent 2100 Bioanalyzer. Large differences in RNA yield and quality among protocols were found. Some protocols that are commonly used for other species did not yield usable RNA from grapevine leaves. The optimum methods were Tris-lithium chloride, which, while relatively time-consuming, gave consistently high yields of quality RNA at very low cost and was suitable for PCR and microarray hybridization, and RNeasy Midi + polyethylene glycol, which rapidly provided high-quality RNA, but with lower yields.

Key words: RNA isolation, leaf, grape, Vitis vinifera

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