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首页> 外文期刊>African Journal of Biotechnology >Modified protocol for RNA isolation from different parts of field-grown jute plant suitable for NGS data generation and quantitative real-time RT-PCR
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Modified protocol for RNA isolation from different parts of field-grown jute plant suitable for NGS data generation and quantitative real-time RT-PCR

机译：适用于NGS数据生成和定量实时RT-PCR的改良方法，可从田间生长的黄麻植物不同部位分离RNA

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摘要

High quality RNA extraction from field-grown jute plants can be difficult due to the presence of complex polyphenolic, polysaccharide and waxes. But isolation of RNA in high quality is a basic need in plant genetics, genomics, transcriptomics, molecular biology and related physiological investigations. Here, cetyl trimethyl ammonium bromide (CTAB) based modified RNA isolation protocol suitable for isolating high quality total RNA from different parts of field-grown jute plants was reported. Modifications come with two extra wash with extraction buffer, residual polysaccharides precipitation with absolute ethanol and potassium acetate during phenol:chloroform:isoamyl alcohol (PCI), intermediate pelleting with lithium chloride followed by dissolving the pellet in sodium dodecyl sulfate (SDS). The 28S/18S ratio and RIN of isolated RNA were greater than 2.0 and 7.3 to 8.8, respectively reveal RNA to be high purity. The isolated RNA can be used directly for subsequent downstream applications including cDNA library construction, reverse transcription polymerase chain reaction (RT-PCR) and RNA-Seq raw data generation without further purification. Moreover, this study showed that this method could also be successfully applied to other polyphenols and polysaccharides rich malvaceous plants.
机译：由于存在复杂的多酚，多糖和蜡，很难从田间种植的黄麻植物中提取高质量的RNA。但是高质量分离RNA是植物遗传学，基因组学，转录组学，分子生物学和相关生理学研究的基本需求。在此，已报道了适用于十六烷基三甲基溴化铵（CTAB）的改良RNA分离方案，适用于从田间生长的黄麻植物不同部位分离高质量的总RNA。进行了两次修饰，分别用萃取缓冲液洗涤，在无水乙醇和乙酸钾在苯酚：氯仿：异戊醇（PCI）期间残留的多糖沉淀，用氯化锂进行中间沉淀，然后将沉淀溶解在十二烷基硫酸钠（SDS）中。分离的RNA的28S / 18S比率和RIN分别大于2.0和7.3至8.8，表明RNA具有高纯度。分离的RNA可直接用于后续下游应用，包括cDNA文库构建，逆转录聚合酶链反应（RT-PCR）和RNA-Seq原始数据生成，而无需进一步纯化。而且，这项研究表明，该方法也可以成功地应用于其他富含多酚和多糖的异常植物。

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