Isolation of RNA from polysaccharide-rich seeds.
Commonly, RNA isolation is the initial step in the study of gene expression analysis and also in the utilization of genes for genetic improvement. However, the recovery of large amounts of RNA with high quality is a difficult process, especially in tissues containing enhanced levels of polysaccharides and other secondary metabolites. Since several procedures for RNA isolation from polysaccharides rich plant tissues have been resulting in poor yields, an effective new protocol is essential for RNA isolation and further analysis. Here, we describe a novel modified technique for isolating total RNA from maturing grains. As a model, we utilized little finger millets, important food staples, which correspond to short duration crops cultivated in varied agro climatic conditions. After isolation, the total RNA was resolved on a denaturing agarose gel, showing more sharp bands of 28S, 18S, and 5S with no degradation. Therefore, the RNA concentration (higher than 1.80) was calculated by spectrophotometry, indicating that RNA is concentrated. Finally, RT-PCR and Northern hybridization confirmed high RNA quality.
Sivakumar
S
S
Centre for Plant Molecular Biology, Tamilnadu Agricultural University, Coimbatore, India.
Franco
O L
OL
Thayumanavan
B
B
D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018556:Crops, Agricultural / Q000737:chemistry; D018076:DNA, Complementary / Q000096:biosynthesis; D004587:Electrophoresis, Agar Gel; D031748:Eleusine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D017343:Genes, Plant; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D011134:Polysaccharides / Q000737:chemistry*; D012333:RNA, Messenger / Q000302:isolation & purification; D018749:RNA, Plant / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D018076:DNA, Complementary; D011134:Polysaccharides; D012333:RNA, Messenger; D018749:RNA, Plant