Hexadecyltrimethylammonium Bromide (CTAB)-based Protocol to Isolate High-Quality RNA in Adequate Quantities for Gene Expression Analyses in Developing Seeds of Lentils (Lens culinaris Medik.)

Abstract

RNA isolation from polysaccharide-and phenolics-rich plant tissues such as developing lentil (Lens culinaris Medik.) seeds is challenging. High-quality RNA is needed in adequate quantities for transcriptome analysis to study seed quality traits. To date, a suitable method to isolate high-quality and -quantity RNA from lentil seeds has not been reported. The objective of this study was to develop a simple and reproducible method to isolate highquantity and -quality RNA from developing lentil seeds for gene expression analysis. Methods based on Trizol™ reagents and phenol:guanidine gave low yields of RNA. A method based on hexadecyltrimethylammonium bromide followed by a lithium chloride precipitation yielded RNA in high quantity (210-260 µg from 200 mg of seeds) and quality (A 260/280 ratio of about 2.2). Isolated RNA was used to study the expression of the granule-bound starch synthase I (GbssI) during lentil seed development by RNA gel blot and quantitative real-time polymerase chain reaction. The expression pattern of the GbssI was similar to that reported for pea GbssI gene.