OPTIMIZATION OF HIGH QUALITY RNA ISOLATION METHOD FROM STEVIA REBAUDIANA BERTONI LEAF FOR Q-PCR ANALYSIS
SAYANTI KUNDU *
UGC Centre for Advanced Study, Department of Botany The University of Burdwan, Burdwan-713104 West Bengal, India
AVISHEK DEY *
UGC Centre for Advanced Study, Department of Botany The University of Burdwan, Burdwan-713104 West Bengal, India
ABHIJIT BANDYOPADHYAY *
UGC Centre for Advanced Study, Department of Botany The University of Burdwan, Burdwan-713104 West Bengal, India
*Author to whom correspondence should be addressed.
Abstract
A suitable method has been optimized to isolate high quality RNA from polyphenols and glycosides rich leaves of Stevia rebaudiana for Q-PCR analysis. RNA isolation protocol was standardized through some modifications of standard guanidinium isothiocyanate – phenol based method, CTAB-LiCl based method and tris - saturated phenol based method. Quality and yield of isolated RNA were assessed through spectrophotometric technique, band profile in 1.5% (w/v) nondenaturing agarose gel electrophoresis and amplification efficiency during Q-PCR analysis. Modification of standard guanidinium isothiocyanate – phenol based RNA isolation method through addition of 2% (w/v) polyvinyl pyrrolidone and 2% (w/v) β-mercaptoethanol during extraction followed by chloroform: isoamyl alcohol (24:1) induced phase separation and precipitation in 8M lithium chloride was found to be best with respect to quality, quantity and downstream application of isolated RNA. The proposed protocol is indeed promising to isolate high quality RNA from secondary metabolites rich S. rebaudiana leaves.
Keywords: RNA isolation, Q-PCR, Stevia rebaudiana