These miRNA mimics are typically from C.elegans, and bear no sequence homology to mammalian miRNAs. The levels of these spike-in controls can therefore be used to normalize for the cumulative experimental error introduced from all downstream procedures, including differences in RNA extraction, reverse transcription, and PCR efficiency. While this strategy is practical to implement, it suffers from a potentially significant drawback in that it cannot compensate for technical variations that take place upstream of the spike-in event, such as might occur during the collection, transport and/or storage of blood/plasma. This is particularly a concern when using samples that have been stored for variable periods, or when comparing results fromdifferentgroups that mayuse different methods for processing and storage of samples. Technical variations associated with these upstream steps are expected to be operator/study specific, and therefore may affect there producibility and interpretation of results. We hypothesized that Proadifen hydrochloride circulating miRNAs with adequate ‘expression’ stability in plasma could be empirically identified without a priori knowledge of function, and these internal reference controls could improve the assessment of disease-related changes in circulating miRNA. Toward this end, we systematically examined the relative plasma level stability of over 1000 different miRNAs across healthy subjects and two very different disease contexts: pulmonary arterial hypertension and septic shock. This study demonstrates the feasibility of identifying circulating miRNA reference controls on a denovo basis, and reveals important new insight into the relative performance of different normalization strategies. Reduced miR-26a levels in PAH could also be revealed by normalization to the miR-142-3p/PJ34 miR-320a pair, and by miR-320a alone, but not miR-1423p alone. This result illustrates the benefit of using multiple reference controls as a means to mitigate the potential biases associated with any single reference control. The effects of less stable reference controls were evaluated by normalizing with miR-16 and miR-328, which served to erode the difference in miR-26a levels between healthy control and PAH subjects.