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A method for isolating total RNA from mature buds and other woody tissues of Vitis vinifera
Year:
2010
Authors :
Halaly, Tamar
;
.
Keren, Alexandra
;
.
Ogrodovitch, Aliza
;
.
Or, Etti
;
.
Rotman, Ariel
;
.
Volume :
Co-Authors:
Acheampong, A.K., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Rotman, A., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Zheng, C., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Keren, A., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Halaly, T., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Crane, O., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Ogrodovitch, A., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Or, E., Department of Horticulture, Agricultural Research Organisation, Volcani Center, Bet Dagan, Israel
Facilitators :
From page:
301
To page:
307
(
Total pages:
7
)
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. © Springer Science+Business Media B.V. 2010.
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DOI :
10.1007/978-90-481-9283-0_20
Article number:
Affiliations:
Database:
Scopus
Publication Type:
Book chapter
;
.
Language:
English
Editors' remarks:
ID:
27703
Last updated date:
02/03/2022 17:27
Creation date:
17/04/2018 00:33
You may also be interested in
Scientific Publication
A method for isolating total RNA from mature buds and other woody tissues of Vitis vinifera
Acheampong, A.K., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Rotman, A., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Zheng, C., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Keren, A., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Halaly, T., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Crane, O., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Ogrodovitch, A., Institute of Plant Sciences, A.R.O. Volcani Center, Bet Dagan, Israel
Or, E., Department of Horticulture, Agricultural Research Organisation, Volcani Center, Bet Dagan, Israel
A method for isolating total RNA from mature buds and other woody tissues of Vitis vinifera
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. © Springer Science+Business Media B.V. 2010.
Scientific Publication
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