Influence of RNA labeling on expression profiling of microRNAs.

Although a number of technical parameters are now being examined to optimize microRNA profiling experiments, it is unknown whether reagent or component changes to the labeling step affect starting RNA requirements or microarray performance. Human brain/lung samples were each labeled in duplicate, at 1.0, 0.5, 0.2, and 0.1 μg of total RNA, by means of two kits that use the same labeling procedure but differ in the reagent composition used to label microRNAs. Statistical measures of reliability and validity were used to evaluate microarray data. Cross-platform confirmation was accomplished using TaqMan microRNA assays. Synthetic microRNA spike-in experiments were also performed to establish the microarray signal dynamic range using the ligation-modified kit. Technical replicate correlations of signal intensity values were high using both kits, but improved with the ligation-modified assay. The drop in detection call sensitivity and miRNA gene list correlations, when using reduced amounts of standard-labeled RNA, was considerably improved with the ligation-modified kit. Microarray signal dynamic range was found to be linear across three orders of magnitude from 4.88 to 5000 attomoles. Thus, optimization of the microRNA labeling reagent can result in at least a 10-fold decrease in microarray total RNA requirements with little compromise to data quality. Clinical investigations bottlenecked by the amount of starting material may use a ligation mix modification strategy to reduce total RNA requirements.