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
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.
Key words: Gene expression
studies, Real-time PCR, RNA isolation, Starch-rich grain, Wheat