Tissue fractionation by hydrostatic pressure cycling technology: the unified sample preparation technique for systems biology studies.
Tissue fractionation by hydrostatic pressure cycling technology: the unified sample preparation technique for systems biology studies.
复制标题
静水压循环技术的组织分离：系统生物学研究的统一样品制备技术。
DOI：
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发表时间：
2008
影响因子：
--
通讯作者：
A. Lazarev
中科院分区：
文献类型：
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作者：
Vera S. Gross;Greta Carlson;Ada T Kwan;G. Smejkal;E. Freeman;A. Ivanov;A. Lazarev
系统生物学研究中的主要瓶颈来自当前样品制备技术的限制。从细胞和组织中提取不同类别的分子通常需要多种相互排斥的样品制备方法，这些方法与珍贵或非常有限的样品的研究不相容。此外，溶解样品组分通常所需的强去污剂和离液剂经常干扰随后的分离和分析。在这里，我们描述了一种快速，无洗涤剂的样品制备技术，允许有效的并行分离和分馏的蛋白质，DNA，RNA和脂质的生物样品，消除了多次重复的需要。该方法依赖于通过静水压力（压力循环技术）对细胞材料的物理破坏和新颖的提取条件的协同组合，以将不同类别的分子溶解和分配到单独的级分中。我们证明了平行回收的蛋白质，脂质，和完整的DNA和RNA，从动物细胞和组织，蛋白质组学，脂质组学和基因组学分析。蛋白质提取物需要最少的净化，并与1D和2D PAGE，液相色谱串联质谱法，和蛋白质印迹法相兼容。脂质馏分已被描绘通过基质辅助激光解吸/电离飞行时间质谱法，无需进一步处理。通过琼脂糖凝胶可视化显示分离的DNA和RNA是完整的，并且通过真实的时间逆转录聚合酶链反应证实完整mRNA的存在。使用这种方法和更传统的提取技术提取的样品的分析和比较揭示了几种蛋白质种类优先提取的新方法。
Major bottlenecks in systems biology studies arise from limitations of current sample preparation techniques. Multiple mutually exclusive sample preparation methods, which are often required to extract distinct classes of molecules from cells and tissues, are incompatible with studies of precious or very limited samples. Moreover, the strong detergents and chaotropic agents commonly required to solubilize sample constituents often interfere with subsequent separation and analysis. Here we describe a rapid, detergent-free sample preparation technique that allows efficient concurrent isolation and fractionation of protein, DNA, RNA, and lipids from biological samples, eliminating the need for multiple replicates. The method relies on a synergistic combination of physical disruption of the cellular material by hydrostatic pressure (pressure cycling technology) and novel extraction conditions to dissolve and partition distinct classes of molecules into separate fractions. We demonstrate parallel recovery of proteins, lipids, and intact DNA and RNA, from animal cells and tissues, for proteomic, lipidomic, and genomic analyses. The protein extracts require minimal cleanup and are compatible with 1D and 2D PAGE, liquid chromatography coupled with tandem mass spectrometry, and Western blotting. The lipid fractions have been profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without further processing. The isolated DNA and RNA were shown to be intact by agarose gel visualization, and the presence of intact mRNA was confirmed by real time reverse transcription polymerase chain reaction. Analysis and comparison of samples extracted using this method and a more traditional extraction technique revealed several protein species preferentially extracted by the new method.
DOI：
10.2741/2258
发表时间：
2007-01-01
期刊：
FRONTIERS IN BIOSCIENCE
影响因子：
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作者：
Han, Xianlin
通讯作者：
Han, Xianlin