A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics.
BACKGROUND
Isolation of pure RNA from woody perennials, especially fruit crops such as grapevine rich in complex secondary metabolites, has remained very challenging. Lack of effective RNA isolation technology has resulted in difficulties in viral diagnosis and discovery as well as studies on many biological processes of these highly important woody plants. It is imperative to develop and refine methodologies with which large amounts of pure nucleic acids can be readily isolated from woody perennials.


METHODS
We compared five commonly used RNA isolation kits in isolating total RNA from twelve species of woody perennials. We made modifications to select RNA isolation systems to simplify and improve their efficiency in RNA isolation. The yield and quality of isolated RNAs were assessed via gel electrophoresis and spectrophotometric measurement. We also performed RT-PCR and RT-qPCR to detect several major viruses from grapevines.


RESULTS
Two of the kits were shown to be the best in both the yield and quality of the isolated RNA from all twelve woody species. Using disposable extraction bags for tissue homogenization not only improved the yield without affecting quality, but also made the RNA isolation technology simpler, less costly, and suitable for adoption by many potential users with facility limitations. This system was successfully applied to a wide range of woody plants, including fruit crops, ornamentals and timber trees. Inclusion of polyvinylpyrrolidone in the extraction buffer drastically improved the performance of the system in isolating total RNA from old grapevine leaves collected later in the season. This modification made our system highly effective in isolating quality RNA from grapevine leaves throughout the entire growing season. We further demonstrated that the resulting nucleic acid preparations are suitable for detection of several major grapevine viruses with RNA or DNA genomes using PCR, RT-PCR and qPCR as well as for assays on plant microRNAs.


CONCLUSIONS
This improved RNA isolation system would have wide applications in viral diagnostics and discovery, studies on gene expression and regulation, transcriptomics, and small RNA biology in grapevines. We believe this system will also be useful in diverse applications pertaining to research on many other woody perennials and recalcitrant plant species.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada. huogen@uoguelph.ca.
Huogen
H
Xiao
Norgen BioTek, Thorald, ON, L2V 4Y6, Canada. wonsik.kim@norgenbiotek.com.
Won-Sik
WS
Kim
Department of Molecular and Cellular Biology, University of Guelph, Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada. bmeng@uoguelph.ca.
Baozhong
B
Meng
D004586:Electrophoresis; D008967:Molecular Biology / Q000379:methods*; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D010944:Plants / Q000821:virology*; D012313:RNA / Q000302:isolation & purification*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013053:Spectrophotometry; D014773:Virology / Q000379:methods*
D012313:RNA