Abstract
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.
Highlights
Lentil (Lens culinaris ssp. culinaris) is a cool-season grain legume cultivated throughout world and in Indian subcontinent providing a vital source of dietary protein in human diets and straw for animal feed
Obtaining high-quality RNA is a crucial requirement in developing genomic resources such as performing gene expression experiments, cDNA library construction, real-time PCR or microarray
Standard methods for RNA extraction (Chomczynski and Sacchi 1987; Salzman et al 1999) could not be used for lentil tissues as they are rich in multiple macromolecular components
Summary
Lentil (Lens culinaris ssp. culinaris) is a cool-season grain legume cultivated throughout world and in Indian subcontinent providing a vital source of dietary protein in human diets and straw for animal feed. Culinaris) is a cool-season grain legume cultivated throughout world and in Indian subcontinent providing a vital source of dietary protein in human diets and straw for animal feed. It is a diploid (2n = 29 = 14), annual flowering self-pollinating crop with a genome size of 4 Gbp (Kaur et al 2011). Lipids associated with proteins and carbohydrates interfere with RNA isolation protocols If these macromolecular contaminants are not removed, they lead to erroneous estimations of RNA quantity and interfere with reverse transcription and PCR, compromising gene expression studies (Koonjul et al 1999). The isolated RNA from all the tissues is amenable for downstream applications such as PCR, RT-PCR and cDNA library construction
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