ABSTRACT
The growing recognition of the importance of miRNAs in cellular regulation has given rise to considerable interest in measuring miRNA levels in biological samples. We discuss here some of the important factors to be considered to ensure accurate, sensitive, and reproducible measurements of miRNA levels, with particular attention to the Agilent miRNA microarray system. We compare and contrast microarray with qPCR measurements, showing that there is in general excellent agreement between Agilent microarray and TaqMan qPCR miRNA measurements, though with some particular exceptions. We discuss methods of assessing RNA quality, and the importance of standardizing sample prep methods to increase reproducibility and avoid some sources of systematic variation. When choosing techniques and transforms to apply to the data, it is important to understand the sensitivity and dynamic range of the measurement. We show examples of dose-response curves for array and qPCR assays. We then discuss some potential sources of measurement bias, and propose some methods of normalizing the raw data which attempt to correct for bias from different sources. Finally, we give a brief overview of methods for analyzing miRNA expression profiling data and some caveats that must be considered.
