An improved method for isolation of high-quality RNA from starch-rich wheat grains

ABSTRACT

Aim: The aim of the present investigation was to develop a quick, easy and reliable method for isolation of RNA from starch rich mature wheat grains; and to check the quality of isolated RNA for downstream applications.Methodology: In the present protocol, highly efficient modified RNA extraction buffer [100 mM Tris (pH 9.0), 150 mM NaCl, 50 mM EDTA, 1.5% sodium dodecyl sulfate (SDS) and 1.5% 2-mercaptoethanol] was used, subsequently followed by TRIzol extraction. Carryover starch was effectively solubilized by adding NaCl before RNA precipitation step. RNA quality was assured by agarose gel electrophoresis, spectrophotometric analysis and quantitative real-time PCR. Results: The problem of co-precipitation of starch along with RNA was resolved effectively. Intact sharp bands of 18S and 28S rRNA on agarose gel confirmed the integrity of isolated RNA. The average A260/A280 ratios ranged from 2.06 to 2.11 and A260/A230 ratio was higher than the respective A260/A280 ratio, indicating high purity of isolated RNA. The isolated RNA was found suitable for gene expression analysis through quantitative real-time PCR. Interpretation: An improved quick, easy and reliable method developed for isolation of high-quality RNA from starch-rich mature wheat grains could be useful for downstream molecular analysis: