Microarray Facility Tübingen, Institute of Human Genetics, University of Tübingen, Tübingen, Germany.
Biotechniques. 2010 May;48(5):371-8. doi: 10.2144/000113421.
Microarrays evolved from a highly specialized technique into a standard molecular biology method that is widely used for whole-genome gene expression profiling. One of the most important aspects of this method is sample identity, that is, whether the expression profile recorded from an array actually derives from the indicated sample. Several potential steps in the protocol exist where a mix-up of samples may occur. With the increasing size of microarray studies, it is important to ensure that each expression profile is assigned to the correct sample. Errors at this level almost certainly lead to erroneous results and can even cause a complete failure of the microarray study. We developed a system that utilizes probes already present on commercially available Affymetrix arrays to unambiguously correlate the recorded expression profile with the input sample RNA. A set of eight spike-in controls were generated, which can be added to sample RNA in different combinations to generate an "on-chip identifier" that passes through the entire array processing protocol and results in a sample-specific hybridization pattern. This pattern can then be used to monitor whether each array was hybridized with the correct sample. The spike-in controls did not have any negative effect on RNA integrity or any detectable influence on the expression values of the remaining probes on the array; therefore, these controls represent an inexpensive and easily adaptable system to guarantee high-quality results from microarray experiments.
微阵列技术从一种高度专业化的技术演变为一种广泛应用于全基因组基因表达谱分析的标准分子生物学方法。该方法最重要的方面之一是样品的身份，即从阵列中记录的表达谱实际上是否来自指定的样品。在方案的几个潜在步骤中，可能会出现样品混淆的情况。随着微阵列研究规模的不断扩大，确保每个表达谱都分配给正确的样品非常重要。在这个层面上的错误几乎肯定会导致错误的结果，甚至可能导致微阵列研究的完全失败。我们开发了一种利用商业上可用的 Affymetrix 芯片上已有的探针的系统，以明确地将记录的表达谱与输入的样品 RNA 相关联。生成了一组 8 个 Spike-in 对照物，可以将其以不同的组合添加到样品 RNA 中，以生成一个“芯片内标识符”，该标识符通过整个芯片处理方案，并产生样品特异性的杂交模式。然后可以使用该模式来监测每个芯片是否与正确的样品杂交。Spike-in 对照物对 RNA 完整性没有任何负面影响，也对芯片上其余探针的表达值没有任何可检测的影响；因此，这些对照物代表了一种廉价且易于适应的系统，可以保证微阵列实验产生高质量的结果。
