The choice of endogenous controls in exosomal microRNA assessments from biofluids

The assessment of differentially expressed microRNAs in patients and healthy controls is important to identify potential tumor biomarkers. Recently, it has been shown that the microRNA levels in exosomes are more correlated with the clinical-pathological variables than vesicle-free microRNAs (miRNAs) in biofluids; therefore, there is an increasing interest in these specific evaluations. However, these measurements can be affected by experimental problems that not always are evaluated and/or by inadequate procedural choices. In particular, exosome isolation and miRNA extraction procedures are crucial to avoid contaminations, and even the choice of the most suitable purity controls is important. Moreover, a stable endogenous RNA should be used for normalization of miRNA expression obtained by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) in order to make these measures comparable among different samples. A rushed choice of the endogenous control can bias study conclusions without revealing inconsistencies. Unfortunately, a few studies systematically identified the best normalizer for their specific experimental context. Instead, sometimes, the normalization procedures were performed in a disputable way or the normalizer choices simply based on the previous literature. Here, we reviewed the studies where the exosomal miRNA profiling was assessed in human biofluids to point out the adopted procedures and the specific endogenous controls chosen for normalization.