Influence of RNA Labeling on Expression Profiling of MicroRNAs

Several technical parameters are now being optimized for microRNA expression profiling experiments, but the finite amount of total RNA available continues to bottleneck clinical investigations. We used microarrays to determine the influence of the labeling reagent on total RNA requirements and data quality. Human brain/lung samples were each labeled in duplicate, at 1.0ug, 0.5ug, 0.2ug, and 0.1ug of total RNA, using two microRNA labeling kits (Genisphere Biotin and Biotin-HSR) that utilize the same labeling procedure but differ in the composition of the labeling reagent used to label the microRNA molecules in the samples. A total of 32 arrays were used for this experiment. Additionally, synthetic microRNA spike-in experiments were also performed to establish the signal dynamic range of the microarray using the Biotin-HSR labeling kit. Two-fold serial dilution samples were prepared that contained from 5000 to 4.88 attomoles of miRNA. A total of 12 arrays were used for this experiment, including a negative control sample.