The effect of column purification on cDNA indirect labelling for microarrays.
The effect of column purification on cDNA indirect labelling for microarrays.
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DOI：
10.1186/1746-4811-3-9
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发表时间：
2007-06-27
期刊：
影响因子：
5.1
通讯作者：
Kiss, John Z
中科院分区：
文献类型：
--
作者：
Molas, M Lia;Kiss, John Z
微阵列重现性的成功取决于标准化程序的性能。自从微阵列技术引入全球基因表达分析以来，不同实验室之间结果的可重复性一直是一个主要问题。造成这种差异的两个主要因素是使用不同的微阵列平台和不同的实验室实践。在本文中，我们解决了后一个问题，如何在一个标签程序的步骤之一的变化影响的cDNA产品之前，微阵列杂交。我们使用一个标准的程序来标记cDNA的微阵列杂交，并采用不同类型的柱层析cDNA纯化。在纯化标记的cDNA后，我们使用Agilent 2100 Bioanalyzer和琼脂糖凝胶电泳来评估标记的cDNA的质量，然后将其杂交到微阵列平台上。由于使用四种不同的柱进行纯化，cDNA图谱（即cDNA片段长度和丰度）存在重大差异。此外，不同的柱具有不同的去除rRNA污染的效率。本研究表明，在这种类型的协议中使用的适当的列必须通过实验确定。最后，我们提出了新的证据，确立了在间接标记过程中检测纯化方法的重要性。我们的研究结果证实了在杂交到微阵列平台上之前的标记程序中评估样品质量的重要性。标准化的柱纯化系统用于标记程序将提高不同实验室之间的微阵列结果的再现性。此外，在微阵列杂交之前实施标记样品的质量控制检查点将防止劣质样品与昂贵的微阵列杂交。
The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays.