Circulating microRNAs: understanding the limits for quantitative measurement by real-time PCR

Ever since the discovery of small, non‐coding RNAs in circulation, microRNAs have gained significant attention as biomarkers or, in some instances, regulators of disease and/or associated complications. Until now, most articles published use real‐time quantitative PCR (qPCR) for assessment of circulating RNAs as microarray‐based methods have low reproducibility.1, 2 Real‐time qPCR technology provides us with a simple tool to efficiently determine the amount of a gene transcript in a given sample. However, the simplicity of this methodology can itself be problematic, as one tends to overlook critical factors that make this technique work. Here, we discuss specific factors that must be considered when performing, presenting, and interpreting miRNA quantitative transcript data. Currently, there are 3 main methods of quantifying circulating miRNAs: real time PCR (qPCR), gene arrays, and sequencing. Although they all have individual benefits, qPCR is the favored approach 3 and will be the focus of this article.