Isolation of high quality RNA from Phyllanthus emblica and its evaluation by downstream applications.
Next generation sequencing is a high-throughput technique widely used for transcriptome profiling. Isolation of high quality RNA is a prerequisite for such large scale transcriptome analysis. Phyllanthus emblica is an important medicinal plant having high amount of metabolites like vitamin C, flavonoids, polyphenolic compounds, tannins, which are responsible for its wondered medicinal properties. High concentration of secondary metabolites like polysaccharides and polyphenols proved to be an obstacle in isolating RNA of good quality. Any compromise with quality of RNA affects the downstream applications and requires extra cleaning steps that further reduce RNA quantity. We have developed a protocol for isolation of high quality RNA from P. embilca. RNA was successfully assessed for downstream applications like reverse transcription polymerase chain reaction, rapid amplification of cDNA ends, mRNA library preparation, and sequencing using HiSeq(™) 2000 sequencing technology. The protocol is simple and can be completed in 4-5 h.
Department of Biotechnology, Panjab University, Chandigarh 160014, India.
Avneesh
A
Kumar
Kashmir
K
Singh
D001205:Ascorbic Acid / Q000032:analysis; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D029842:Phyllanthus emblica / Q000737:chemistry / Q000235:genetics*; D010946:Plants, Medicinal / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D059808:Polyphenols / Q000032:analysis; D011134:Polysaccharides / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA
D018076:DNA, Complementary; D059808:Polyphenols; D011134:Polysaccharides; D012333:RNA, Messenger; D018749:RNA, Plant; D001205:Ascorbic Acid