Department of Biotechnology, Panjab University, Chandigarh 160014, India.
Mol Biotechnol. 2012 Nov;52(3):269-75. doi: 10.1007/s12033-011-9492-5.
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.
下一代测序是一种高通量技术，广泛用于转录组谱分析。高质量 RNA 的分离是进行此类大规模转录组分析的前提条件。余甘子是一种重要的药用植物，其代谢物如维生素 C、类黄酮、多酚化合物、单宁含量很高，这也是其具有神奇药用特性的原因。高浓度的次生代谢物，如多糖和多酚，被证明是分离高质量 RNA 的障碍。任何对 RNA 质量的妥协都会影响下游应用，并需要额外的清洁步骤，进一步减少 RNA 的数量。我们已经开发了一种从余甘子中分离高质量 RNA 的方案。成功地评估了 RNA 用于下游应用，如逆转录聚合酶链反应、cDNA 末端快速扩增、mRNA 文库制备和使用 HiSeq(™) 2000 测序技术进行测序。该方案简单，可在 4-5 小时内完成。
