Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry.
Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry.
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使用离子对反相色谱和质谱法对DSRNA的纯化和表征。
DOI：
10.1016/j.chroma.2016.12.062
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发表时间：
2017-02-10
期刊：
影响因子：
--
通讯作者：
Dickman MJ
中科院分区：
文献类型：
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作者：
Nwokeoji AO;Kung AW;Kilby PM;Portwood DE;Dickman MJ
在一步方案中快速纯化dsRNA。高通量纯化和分析多种dsRNA。开发了IP RP HPLC用于dsRNA的快速、高分辨率分析。开发了一种利用RNase T1进行dsRNA的RNase质量作图的新方法。RNA干扰为广泛的生物系统提供了有价值的见解，并且是分析基因功能的有力工具。利用这一途径阻断特定基因靶点的表达，为开发新的基于RNAi的昆虫管理策略提供了相当大的希望。此外，RNAi在控制农业害虫以及预防益虫疾病方面具有广泛的未来潜在应用。通过体外转录或在用于RNA干扰应用的细菌系统中合成大量dsRNA的潜力已经产生了对用于快速提取、纯化和分析dsRNA的高通量分析工具的开发和应用的显著需求。在这里，我们开发了分析方法，能够从细菌细胞的相关杂质中快速纯化dsRNA，并结合下游分析。我们已经优化了TRIzol提取结合一步方案，以去除污染的DNA和ssRNA，在高盐条件下使用RNase T1/DNase I消化结合固相提取纯化dsRNA。此外，我们还利用并开发了IP RP HPLC，用于dsRNA的快速、高分辨率分析。此外，我们已经优化了RNA酶A对dsRNA的碱基特异性切割，并开发了一种利用RNA酶T1进行RNA酶质量作图的新方法，以使用液相色谱与质谱联用进一步鉴定dsRNA。
rapid purification of dsRNA in a single step protocol. high throughput purification and analysis of a wide range of dsRNAs. developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. developed a novel method utilising RNase T1 for RNase mass mapping of dsRNA. RNA interference has provided valuable insight into a wide range of biological systems and is a powerful tool for the analysis of gene function. The exploitation of this pathway to block the expression of specific gene targets holds considerable promise for the development of novel RNAi-based insect management strategies. In addition, there are a wide number of future potential applications of RNAi to control agricultural insect pests as well as its use for prevention of diseases in beneficial insects. The potential to synthesise large quantities of dsRNA by in-vitro transcription or in bacterial systems for RNA interference applications has generated significant demand for the development and application of high throughput analytical tools for the rapid extraction, purification and analysis of dsRNA. Here we have developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised TRIzol extractions in conjunction with a single step protocol to remove contaminating DNA and ssRNA, using RNase T1/DNase I digestion under high-salt conditions in combination with solid phase extraction to purify the dsRNA. In addition, we have utilised and developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. Furthermore, we have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry.