title: Isolation of high quality RNA from pistachio (Pistacia vera L.) and other woody plants high in secondary metabolites.
abstract: The quality and quantity of RNA are critical for successful downstream transcriptome-based studies such as microarrays and RNA sequencing (RNA-Seq). RNA isolation from woody plants, such as Pistacia vera, with very high amounts of polyphenols and polysaccharides is an enormous challenge. Here, we describe a highly efficient protocol that overcomes the limitations posed by poor quality and low yield of isolated RNA from pistachio and various recalcitrant woody plants. The key factors that resulted in a yield of 150 μg of high quality RNA per 200 mg of plant tissue include the elimination of phenol from the extraction buffer, raising the concentration of β-mercaptoethanol, long time incubation at 65 °C, and nucleic acid precipitation with optimized volume of NaCl and isopropyl alcohol. Also, the A260/A280 and A260/A230 of extracted RNA were about 1.9-2.1and 2.2-2.3, respectively, revealing the high purity. Since the isolated RNA passed highly stringent quality control standards for sensitive reactions, including RNA sequencing and real-time PCR, it can be considered as a reliable and cost-effective method for RNA extraction from woody plants.
authors.affiliation: Plant Biotechnology Department, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Moazzam Jazi
authors.affiliation: Environmental and Industrial Biotechnology Department, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Saideh
authors.initials: S
authors.lastname: Rajaei
authors.affiliation: Plant Biotechnology Department, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Seyed Mahdi
authors.initials: SM
authors.lastname: Seyedi
keywords: Pistacia vera; Polyphenols; RNA extraction; RNA-sequencing; Real-time PCR; Tree