A small-scale RNA isolation protocol useful for high-throughput extractions from recalcitrant plants

Abstract

Many plants indigenous to South Africa are rich in secondary and oxidizing compounds such as pigments, complex polysaccharides and polyphenols. This makes isolation of high quality RNA for analysis of gene expression difficult. Here we describe a cost-effective isolation protocol suitable for RNA extraction from recalcitrant plant species. This method uses small amounts of tissue, so is useful when material is limited, and is easy to process large numbers of samples at once. We have used the method successfully with mature leaves of Protea hybrid ‘Sylvia’, and species P. repens, Leucospermum hybrid ‘Succession’, resurrection plants Xerophyta humilis and Craterostigma pumilum, and mature needles of Pine (Pinus radiata). RNA was analyzed spectrophotometrically and was found to be of high purity with low levels of contaminating compounds. Electrophoretic analyses on denaturing formaldehyde agarose gels and an Agilent 2100 Bioanalyzer confirmed the presence of RNA of high integrity. This is the first description of plant RNA integrity number (RIN) values for these plants using the algorithm designed for analyses of plant RNA containing multiple ribosomal bands. The RNA could successfully be used for reverse transcription and gene amplification.