Isolation of high-quality RNA from recalcitrant leaves of variegated and resurrection plants

Abstract

Resurrection plant Ramonda serbica is a suitable model to investigate mechanisms of desiccation tolerance, while variegated Pelargonium zonale has been proven to serve as an excellent model for the metabolite allocation between sink tissue and source tissue within the same organ. However, the genomes of these plants are still not sequenced, limiting their application in molecular studies. To investigate the transcript abundance by next-generation sequencing, high-quality RNA input is required. Leaves of both P. zonale and R. serbica are rich in polyphenols that interfere with high-quality RNA extraction by common protocols. Moreover, low water content and high amount of sugars and other osmoprotectants in desiccated R. serbica leaves present the additional challenge in total RNA extraction. Here, we evaluated and compared several already established TRIzol- and CTAB-based protocols aiming to develop the efficient, simple and low-cost methods for the extraction of the satisfactory yield RNA of great purity and integrity, required for the construction of high-quality cDNA libraries. Our results show that the CTAB-based protocol (i.e. CTAB 1b) enabled the extraction of high-quality RNA from photosynthetically active and non-photosynthetically active leaf sectors of P. zonale, with high RIN values. On the other hand, TRIzol-based protocol provided a high RNA yield with low contamination and high RNA integrity even in desiccated leaves of R. serbica. We envisage that the proposed protocol would be suitable for the RNA extractions from other desiccated organs (e.g. seeds, grains, pollen grains).

Keywords: Desiccated leaves, polyphenols; Ramonda serbica; Recalcitrant plant material; Variegated Pelargonium zonale.

Fig. 1

Schematic workflow of three protocols applied for total RNA extraction: TRIzol-based protocol; CTAB 1a and CTAB 1b protocols; and CTAB 2 protocol (see “Materials and methods”). The total RNA was extracted from P. zonale (green—G and white—W leaf sectors) and R. serbica hydrated (HL) and dehydrated leaves (DL). Total RNA samples (1 µg of RNA was loaded) were analysed on 2% agarose gel stained with ethidium bromide. The ladder (L, GeneRuler 1 kb DNA Ladder, Thermo Fisher Scientific) is reported in the first lane. One asterisk and two asterisks indicate the 25S and 18S units of rRNA, respectively

Fig. 2

Digital gel image from the Agilent 2100 BioAnalyzer system showing the representative total RNA isolated from P. zonale (green—G and white—W leaf sectors) by CTAB 1b protocol, and from hydrated (H) and dehydrated (D) R. serbica leaves by TRIzol-based protocol. The ladder (L) is reported in the first lane. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.)