Recent studies have also shown that vesicle-encapsulated miRNAs can be shuttled between cells and modulate gene expression, exerting control over physiological functions in the recipient cells.Thus, beyond their practical application as biomarkers ,circulating miRNAs may also be involved in intercellular signaling. The detection of small Isovaleramide alterations in plasma miRNA levels is challenging because of their relatively low abundance in circulation. Total RNA extracted from plasma is often at or below the limit of detection of UV spectrophotometry, which precludes standardization by RNA mass, and quality control filtering by RNA purity and integrity prior to downstream RT-qPCR measurements. Therefore, stringent normalization of plasma miRNA levels is critical to improve the reproducibility of results, by removing nonbiological technical variations that might otherwise obscure or exaggerate the underlying biological variations of interest. However, there is currently no consensus on how best to normalize plasma miRNA levels, as reflected by the many different methods that have been employed. Classical small RNA reference controls exhibit stable expression levels in cells because of their fundamental “housekeeping” functions, but these RNAs are not routinely detectable in plasma. Moreover, because the biological functions of circulating miRNA have yet to be elucidated, it remains unclear whetherany circulating miRNAs have prototypical housekeeping functions, or would be present at sufficiently stable levels to serve as effective reference controls. Nevertheless, the identification of circulating reference controls represents an important unmet need if circulating miRNA are to JNJ-7777120 advance as robust biomarkers. A conceptual framework necessary to identify and validate circulating reference controls on a denovo basis has not been clearly defined in this emerging field. Therefore, alternative strategies such as the external control, continue to be widely used. This method involves the use of one or more synthetic miRNA mimics that are spiked into each plasma sample at affixed concentration, just after denaturation of endogenous plasma ribonucleases.