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Isolation of High Quality RNA from Cereal Seeds Containing High Levels of Starch
Author(s) -
Wang Guifeng,
Wang Gang,
Zhang Xiaowei,
Wang Fang,
Song Rentao
Publication year - 2011
Publication title -
phytochemical analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 72
eISSN - 1099-1565
pISSN - 0958-0344
DOI - 10.1002/pca.1337
Subject(s) - trizol , chemistry , rna , rna extraction , lysis buffer , starch , polysaccharide , chromatography , lysis , food science , complementary dna , hydrolysis , dna , biochemistry , gel electrophoresis , sodium dodecyl sulfate , gene
Cereals are an important source of food, feed and fuel with a rapidly increasing global demand. However, cereal seeds contain high levels of starch and polysaccharides, making the isolation of high quality RNA extremely difficult. Objective To develop a novel method for extracting high quality total RNA from various starch‐ and polysaccharides‐rich cereal seeds, such as maize, rice, sorghum and wheat. Methodology We developed a modified sodium dodecyl sulphate (SDS)/TRIzol method. The combined use of a Tris buffer (pH 9.0) and SDS before TRIzol extraction effectively resolved the problem of seed homogenate solidification in such a buffer. A high concentration of SDS was used separately, not only to promote cell lysis but also to effectively dissolve seed sample containing high levels of starch. Moreover, acid phenol saturated with 0.1 m citrate buffer (pH 4.3) was used to separate RNA from DNAs, proteins and high levels of starch. This rapid protocol was compared with other RNA isolation methods preferentially used for plants rich in polysaccharides and secondary metabolites. Results Gel electrophoresis analysis indicated that the extracted total RNA had good integrity without apparent DNA contamination. Furthermore, an A 260/280 ratio of approximately 2.0, an A 260/230 ratio of more than 2.0 and RIN values of more than 8.6 indicated that the isolated RNA was of high purity. The isolated RNA was suitable for subsequent molecular manipulations, such as reverse‐transcription polymerase chain reaction (PCR), rapid amplification of cDNA ends (RACE) and real‐time PCR. Conclusion The study has described an easy, efficient and highly reproducible method for RNA isolation from various cereal seeds. Copyright © 2011 John Wiley & Sons, Ltd.