An efficient protocol for isolation of inhibitor-free nucleic acids even from recalcitrant plants.
For fast and easy isolation of inhibitor-free genomic DNA even from the toughest plant leaf samples, including those high in polyphenols and polysaccharides, a protocol has been developed. To prevent the solubility of polysaccharides in the DNA extract, high salt concentration (1.4 M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) was used for the removal of polyphenols as polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by proteinase K and removed by centrifugation from plant extracts during the isolation process resulting in pure DNA and RNA ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA and RNA isolated from leaves and roots of recalcitrant plants which was free from contamination and color. The average yields of total RNA from roots and shoot of Betula and Grape ranged from 285 to 364 ng/µl with A260/A280 between 1.9 and 2.08. The RNA isolated with this protocol was verified to be suitable for PCR, quantitative real-time PCR, semi-quantitative reverse transcription polymerase chain reaction, cDNA synthesis and expression analysis. This protocol shown here is reproducible and can be used for a broad spectrum of plant species which have polyphenols and polysaccharide compounds.
Department of Biotechnology, Faculty of Agricultural Sciences, University of Guilan, P.O. Box: 41635-1314, Rasht, Iran.
Mohammad Hossein
MH
Rezadoost
Department of Biotechnology, Faculty of Agricultural Sciences, University of Guilan, P.O. Box: 41635-1314, Rasht, Iran.
Mojtaba
M
Kordrostami
Department of Biotechnology, Faculty of Agricultural Sciences, University of Guilan, P.O. Box: 41635-1314, Rasht, Iran. kumleh@guilan.ac.ir.
Hassan Hassani
HH
Kumleh
Betula pendula; DNA isolation; Polyphenol; Polysaccharides; RNA isolation; Vitis vinifera