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Microchip-Based Solid-Phase Purification of RNA from Biological Samples

机译:基于微芯片的生物样品RNA固相纯化

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摘要

Having previously detailed a method for chip-based extraction of DNA (Anal. Chem. 2003, 75, 1880-1886.), we describe here a microchip-based solid-phase extraction method for purification of RNA from biological samples is demonstrated. The method involves the use of silica beads as a solid phase, and the capacity of the device containing silica beads for RNA, RNA in the presence of protein, and DNA was determined. The capacity of the device for RNA binding in the presence of protein is 360 ng, which demonstrates sufficient capacity of the device for complete genetic analysis. An extraction of RNA can be performed on the device in as few as approx9 min (analytical time), a time comparable to that of a commercial extraction method, but with less reagent consumption. The microchip-based extraction is also performed in a closed system, unlike the commercial extraction method, which provides the advantage of decreased opportunity for the introduction of RNases and contaminants-essential for the sensitive RNA-based analyses presented in this work. RNA purified using the device was shown to be amplifiable using reverse transcription PCR (RT-PCR), allowing for translation of the method to the purification and subsequent amplification of biological samples. RNA was purified using the microchip-based method from neat semen, a mock semen stain, and cultured cells from a common pediatric cancer, alveolar rhabdomyosarcoma.
机译:先前已经详细介绍了基于芯片的DNA提取方法(Anal。Chem。2003,75,1880-1886。),在此我们描述了一种基于微芯片的固相提取方法,用于从生物样品中纯化RNA。该方法涉及使用二氧化硅珠粒作为固相,并测定了含有二氧化硅珠粒的装置对RNA,蛋白质和DNA存在下RNA的容量。在蛋白质存在下,该装置用于RNA结合的能力为360 ng,这表明该装置具有足够的能力来进行完整的遗传分析。 RNA的提取可以在大约9分钟(分析时间)内在设备上进行,该时间与商业提取方法相当,但是试剂消耗较少。与商业提取方法不同,基于微芯片的提取也可以在封闭系统中进行,商业提取方法的优点是减少了引入RNase和污染物的机会,这对于本研究中基于敏感RNA的分析至关重要。使用逆转录PCR(RT-PCR)显示使用该设备纯化的RNA可扩增,从而可将该方法翻译为纯化方法,并随后扩增生物样品。使用基于微芯片的方法从纯精液,模拟精液染色以及来自普通儿科癌症肺泡横纹肌肉瘤的培养细胞中纯化RNA。

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