Abstract
Toughness of plant materials and their high secondary plant metabolites, polysaccharides and proteins that bind to and/or co-precipitate with the RNA, are some of the major constraints when extracting total RNA from woody tissues such as mature buds and woody stems. Here, we detail an efficient method for isolating total RNA from woody tissues of grapes. RNA was extracted with high ionic strength buffer at 65°C. Proteins were denatured, and secondary metabolites removed by repeated phenol:chloroform:isoamyl alcohol extractions. The RNA was separated from the DNA by selective precipitation with lithium chloride (LiCl) solution. Though the procedure is laborious and time-consuming, the yield and quality of the RNA extracted were higher compared to other conventional extraction protocols. Yield and purity were spectrophotometrically monitored by UV absorbance (A260/A280 and A260/A230). The yield was about 180 μg total RNA per gram of tissue, and the A260/A280 and A260/A230 absorbance ratios were greater than 2.0. Standard reverse transcription PCR (RT-PCR) yielded 3.5 kb products from RNA isolated by this protocol. Isolated RNA has also been applied in other molecular application such as real-time PCR, Northern blot, dot blot and gene expression studies using Affymetrix Chips.
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Abbreviations
- DEPC:
-
Diethyl pyrocarbonate
- CTAB:
-
Cetyltrimethyl ammonium bromide
- PVP:
-
Polyvinylpyrrolidone
- EDTA:
-
Ethylenediaminetetraacetic acid
- SDS:
-
Sodium dodecyl sulfate
- CIA:
-
Chloroform:Isoamyl alcohol
- PCIA:
-
Phenol:Chloroform:Isoamyl alcohol
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Acheampong, A.K. et al. (2010). A Method for Isolating Total RNA from Mature Buds and Other Woody Tissues of Vitis Vinifera . In: Delrot, S., Medrano, H., Or, E., Bavaresco, L., Grando, S. (eds) Methodologies and Results in Grapevine Research. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9283-0_20
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DOI: https://doi.org/10.1007/978-90-481-9283-0_20
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