Optimization of total RNA extraction protocol from Ziarat Juniper (Juniperus excelsa M.Bieb)

Abstract

Ziarat Juniper (Juniperus excelsa) forest, in Balochistan Province, has importance because of the second largest Juniper forest in the world and one of the oldest slow growing Juniper trees of the world having trees as old as 2500 to 3500 years. Total RNA extraction from Ziarat Juniper will be the entrance of transcriptome world to reveal functional genome of this important plant (Juniperus excelsa). But its high phenolic compounds and polysaccharides contents cause rapidly bind or co-precipitate with RNA during RNA extraction. So standard protocols for RNA extraction do not produce high quality total RNA. In this study, an optimized total RNA extraction protocol based on used of CTAB (Cetyl Trimethyl Ammonium Bromide) PVP (Polyvinyl Pyrrolidone) and 2-mercaptoethanol, in the extraction buffer and an extraction with organic solvents (Phenol and Chloroform) is described for Juniperus excelsa shoot. Agarose gel electrophoresis clearly showed 28S and 18S ribosomal RNA bands and produced RNA with high yield (1.5-2.0 µg/µl) and high quality (1.9-2.0 A260/280 ratio). Thus this protocol for RNA extraction is suitable for molecular analysis of Juniperus excelsa.