Isolation of high-quality nucleic acids from Cistus creticus ssp. creticus and other medicinal plants

Abstract

Leaves of Cistus creticus ssp. creticus (Cistaceae) EDTA, 1.5% (w/v) sodium dodecyl sulfate, 1% (w/v) secrete several labdane-type diterpenes, which display either cytotoxic activity against a number of human leukemic cell lines or antibacterial and antifungal activity [1,2]. Considering the characteristics and possible future applications of these metabolites it seemed worthwhile to study their biosynthesis and metabolism at the molecular level. Thus, it was essential to establish methods for nucleic acid extraction and puriWcation from C. creticus tissues. Cistus species contain high amounts of metabolites that interfer with nucleic acid isolation, such as terpenoids, polyphenols, Xavonoids, Xavonoid aglycones, and glycosides, resin, and thick waxy cuticle covering the aerial parts’ epidermis and especially the leaves [3,4]. As a consequence of these characteristics, several published methods or kits for nucleic acid isolation (e.g., Nucleospin RNA plant kit; Macherey–Nagel, Duren, Germany) [5–10] that were applied to this plant had unsatisfactory results. The only method that gave good-quality total RNA was the one described in Loulakakis et al. [8] but the yield was relatively low (data not shown). In this report, we describe optimized protocols that yield large amounts of highquality genomic DNA and total RNA from C. creticus tissues. The described methods were appropriate for nucleic acid isolation independently of the sampling period, plant age, or plant cultivation method and worked eYciently for other medicinal plant species also. The total RNA extraction procedure is as follows: I Grind 1 g of leaf tissue in Wne powder with mortarl and pestle in liquid nitrogen and place it in a sterile 50-ml tube with 20 ml prechilled extraction buVer [200 mM Tris–HCl, pH 8.5, 300 mM LiCl, 10 mM