Department of Oncology, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge, UK.
BMC Genomics. 2010 Oct 6;11:540. doi: 10.1186/1471-2164-11-540.
The demands of microarray expression technologies for quantities of RNA place a limit on the questions they can address. As a consequence, the RNA requirements have reduced over time as technologies have improved. In this paper we investigate the costs of reducing the starting quantity of RNA for the Illumina BeadArray platform. This we do via a dilution data set generated from two reference RNA sources that have become the standard for investigations into microarray and sequencing technologies.
We find that the starting quantity of RNA has an effect on observed intensities despite the fact that the quantity of cRNA being hybridized remains constant. We see a loss of sensitivity when using lower quantities of RNA, but no great rise in the false positive rate. Even with 10 ng of starting RNA, the positive results are reliable although many differentially expressed genes are missed. We see that there is some scope for combining data from samples that have contributed differing quantities of RNA, but note also that sample sizes should increase to compensate for the loss of signal-to-noise when using low quantities of starting RNA.
The BeadArray platform maintains a low false discovery rate even when small amounts of starting RNA are used. In contrast, the sensitivity of the platform drops off noticeably over the same range. Thus, those conducting experiments should not opt for low quantities of starting RNA without consideration of the costs of doing so. The implications for experimental design, and the integration of data from different starting quantities, are complex.
微阵列表达技术对 RNA 数量的要求限制了它们能够解决的问题。因此，随着技术的不断改进，RNA 的需求量逐渐减少。在本文中，我们研究了降低 Illumina BeadArray 平台起始 RNA 量的成本。我们通过从两个已成为微阵列和测序技术研究标准的参考 RNA 源生成的稀释数据集来实现这一点。
尽管杂交的 cRNA 数量保持不变，但我们发现起始 RNA 量对观察到的强度有影响。当使用较少的 RNA 时，我们会发现灵敏度下降，但假阳性率不会大幅上升。即使使用 10ng 的起始 RNA，阳性结果也是可靠的，尽管许多差异表达基因被遗漏。我们看到，对于贡献了不同 RNA 量的样本，结合数据有一定的空间，但也注意到，当使用低起始 RNA 量时，为了补偿信号噪声的损失，样本量应该增加。
即使使用少量起始 RNA，BeadArray 平台也能保持较低的假发现率。相比之下，平台的灵敏度在同一范围内明显下降。因此，那些进行实验的人不应该在不考虑这样做的成本的情况下选择低起始 RNA 量。实验设计和不同起始量数据的整合具有复杂性。
