Microchip-Based Solid-Phase Purification of RNA from Biological Samples
Microchip-Based Solid-Phase Purification of RNA from Biological Samples
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DOI：
10.1021/ac8011945
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发表时间：
2008-11-15
影响因子：
7.4
通讯作者：
Landers, James P.
中科院分区：
文献类型：
--
作者：
Hagan, Kristin A.;Bienvenue, Joan M.;Landers, James P.
先前已经详细描述了基于芯片的DNA提取方法（Anal. 2003，75，1880-1886），我们在此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作为固相，并测定含有二氧化硅珠的装置对RNA、蛋白质存在下的RNA和DNA的能力。该装置在蛋白质存在下的RNA结合能力为360 ng，这表明该装置具有足够的能力进行完整的遗传分析。RNA的提取可以在该装置上进行，时间短至9分钟（分析时间），与商业提取方法的时间相当，但试剂消耗较少。基于微芯片的提取也是在封闭系统中进行的，与商业提取方法不同，这提供了减少引入RNA酶和污染物的机会的优点，这对于本工作中提出的基于RNA的灵敏分析是必不可少的。使用该装置纯化的RNA显示出使用逆转录PCR（RT-PCR）是可扩增的，允许将该方法转化为生物样品的纯化和随后的扩增。使用基于微芯片的方法从纯精液、模拟精液染色和来自常见儿科癌症腺泡状横纹肌肉瘤的培养细胞中纯化RNA。
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 similar to 9 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 ampliflable 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.