The cost of reducing starting RNA quantity for Illumina BeadArrays: A bead-level dilution experiment
1 Department of Oncology, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK
2 Cancer Research UK - CRI, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK
3 Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, Parkville Victoria 3052, Australia
BMC Genomics 2010, 11:540 doi:10.1186/1471-2164-11-540Published: 6 October 2010
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.