Giorgio Gambino1, Irene Perrone, Ivana Gribaudo. 1. Plant Virology Institute CNR, Grugliasco Unit. Via L. da Vinci 44, I-10095 Grugliasco-TO, Italy. g.gambino@ivv.cnr.it
Abstract
INTRODUCTION: RNA quality and integrity are critical for many studies in plant molecular biology. High-quality RNA extraction from grapevine and other woody plants is problematic due to the presence of polysaccharides, polyphenolics and other compounds that bind or co-precipitate with the RNA. OBJECTIVE: To develop an optimised cetyltrimethylammonium bromide (CTAB)-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from polysaccharide-rich tissues of several plants. METHODOLOGY: Several changes were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4 degrees C, the sample weight was decreased and the concentrations of PVP-40 and LiCl were increased reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with six different RNA extraction methods from three grapevine tissues, namely, in vitro plantlets, and leaves and mature canes from actively growing field vines. RESULTS: The rapid CTAB method gave high-quality RNA in only 3 h at low cost with efficiency equal to or higher than that obtained with other time-consuming and expensive protocols. The procedure was applied to RNA extraction from other grapevine tissues and other woody species including olive, lemon, poplar, chestnut, apple, pear, peach, cherry, apricot, plum and kiwi fruit. RNA of high quality could be isolated from all tissues and from all species. CONCLUSION: The study has shown that the improvement of a CTAB-based protocol allows the rapid isolation of high-quality RNA from grapevine and many woody species.
INTRODUCTION: RNA quality and integrity are critical for many studies in plant molecular biology. High-quality RNA extraction from grapevine and other woody plants is problematic due to the presence of polysaccharides, polyphenolics and other compounds that bind or co-precipitate with the RNA. OBJECTIVE: To develop an optimised cetyltrimethylammonium bromide (CTAB)-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from polysaccharide-rich tissues of several plants. METHODOLOGY: Several changes were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4 degrees C, the sample weight was decreased and the concentrations of PVP-40 and LiCl were increased reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with six different RNA extraction methods from three grapevine tissues, namely, in vitro plantlets, and leaves and mature canes from actively growing field vines. RESULTS: The rapid CTAB method gave high-quality RNA in only 3 h at low cost with efficiency equal to or higher than that obtained with other time-consuming and expensive protocols. The procedure was applied to RNA extraction from other grapevine tissues and other woody species including olive, lemon, poplar, chestnut, apple, pear, peach, cherry, apricot, plum and kiwi fruit. RNA of high quality could be isolated from all tissues and from all species. CONCLUSION: The study has shown that the improvement of a CTAB-based protocol allows the rapid isolation of high-quality RNA from grapevine and many woody species.
Authors: Christelle Lacroix; Kurra Renner; Ellen Cole; Eric W Seabloom; Elizabeth T Borer; Carolyn M Malmstrom Journal: Appl Environ Microbiol Date: 2016-01-15 Impact factor: 4.792
Authors: Mona B Damaj; Phillip D Beremand; Marco T Buenrostro-Nava; Beth Riedel; Joe J Molina; Siva P Kumpatla; Terry L Thomas; T Erik Mirkov Journal: Int J Plant Genomics Date: 2010-01-27