Evaluation of a New High-Dimensional miRNA Profiling Platform

We evaluated a new miRNA profiling platform that utilizes Illumina’s existing robust DASL chemistry as the basis for the assay. Using total RNA from five colon cancer patients and four cell lines, we evaluated the reproducibility of miRNA expression levels across replicates and with varying amounts of input RNA. The beta test version was comprised of 735 miRNA targets of Illumina’s miRNA profiling application. The study used four SAMs to assess variability due to separate total RNA extractions, technical replicates of each extraction and varying total RNA input. Total RNA extracted from five patient samples (IDs 45, 165, 565, 919, 133) and RNA from four cell lines (HeLa, PC3, 293 and MCF-7) supplied by Illumina, was evaluated on each of 4 SAMs. SAM 1 was hybridized in week one, SAMs 2 and 3 were hybridized in week two and SAM 4 (with cell line RNA only) was hybridized in week three of the study. Sample allocation on SAM 1 was designed to assess reproducibility between two separate total RNA extractions, between duplicate samples (technical replicates) and between two input RNA amounts. Sample allocation on SAM 2 was designed to assess technical reproducibility and the effect of six input levels of total RNA. Sample allocation on SAM 3 was designed to assess technical replication. The four cell lines, but none of the 5 patient RNA, were run on a fourth SAM . Reproducibility across all four SAMs was also assessed.