High quality RNA isolation from ployphenol-, polysaccharide- and protein-rich tissues of lentil (Lens culinaris).
Current RNA isolation methods have limitations in their ability to yield good quality and quantity of RNA from plants that have high content of phenols, polysaccharides and storage proteins. Existing methods also do not eliminate accompanying chromosomal DNA in RNA preparation that causes false positives in gene expression studies. Standard isolation technique was modified for rapid and quick extraction of RNA, and lentil tissue most appropriate to extract good quality RNA was determined. The concentration of the phenol blocker polyvinylpyrrolidone in the extraction buffer was determined, DNase I was added to eliminate chromosomal DNA and the timing of this step was optimized. RNA up to 568 μg of RNA from 1 g of tissue was isolated from four different tissues of lentil in less than half the time typically required by reported methods. The method avoids the use of toxic phenol-chloroform, hazardous guanidinium thiocyanate (GTC) and laborious CsCl ultracentrifugation. Absorbance A260/A280 ratio of 1.9 and A260/A230 ratio of 2.7 reveal RNA to be of high purity. Modified method yielded RNA that was free from contaminants and suitable for RT-PCR and cDNA library construction.
NRC on Plant Biotechnology, IARI, LBS Building, PUSA Campus, New Delhi, 110012, India. prasanta01@yahoo.com.
Prasanta K
PK
Dash
Gene library; Legumes; Lentil; Lipids; PCR analysis; Polyphenols; Polysaccharide; RNA isolation; RT-PCR analysis