An efficient and effective RNA extraction protocol for ferns

Abstract

Premise: The extraction of high-quality RNA is the critical first step for the analysis of gene expression and gene space. This remains particularly challenging in plants, and especially in ferns, where the disruption of the cell wall and separation of organic compounds from nucleic acids is not trivial.

Methods: We developed a cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocol that consistently performs well across a large phylogenetic breadth of ferns-a lineage of plants high in secondary compounds-and in an array of tissue types. Two alternative options (precipitation vs. clean-up without intermediate precipitation) are presented, both of which yield high-quality RNA extracts with optical density (OD) ratios of OD 260/280 = 1.9-2.1 and OD 260/230 > 1.6, and RNA integrity numbers >7.

Conclusions: This study presents an efficient protocol for the extraction of high-quality RNA from multiple tissues and across the fern phylogeny, a clade of plants that still lags behind other major lineages in the development of genomic resources. We hope that this method can be used to help facilitate the closing of this gap.

Keywords: RNA extraction; cetyltrimetylammonium bromide (CTAB); ferns; secondary compounds.

Figure 1

Integrity and size of RNA extracted from mature sterile leaflets (A) and roots (B) of Lygodium microphyllum on the Agilent 5400 Fragment Analyzer. rRNAs are largely intact in both samples, no degradation is shown in the lower fragment sizes, and no genomic DNA contamination is present. Major nuclear rRNA peaks (18S around 1.8 kbp and 25S around 3.4 kbp) are identified with black arrows; additional peaks in the samples are representative of organellar rRNAs, which can lead to artificially decreased RIN values. LM, lower marker; nt, nucleotides; RFU, relative fluorescence; RIN, RNA integrity number.