An improved method for isolation of high-quality RNA from starch-rich wheat grains
Date
2020
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Journal of Environmental Biology
Abstract
Aim :
Methodology :
Results :
Interpretation :
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.
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.
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.
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
Description
Keywords
Gene expression studies, Real-time PCR, RNA isolation, Starch-rich grain, Wheat