Normalization using spike-in cel-miR-39 was in this case sufficient to reveal a trend toward the underlying biological difference in miR-26a levels, possibly because the variation between subjects was already relatively low to begin with. In a larger cohort of PAH patients and healthy controls, the lower miR-26a levels in PAH patients was apparent even in raw data, while normalization with cel-miR-39 or the miR-142-3p/ miR-320a pair served to accentuate this difference; however, the use of the latter internal reference controls was associated with a higher level of significance compared to the former external control. A significantly lower plasma level of let-7g was also evident in PAH patients after MCR or internal reference control normalization, but this was again obscured in the non-normalized raw data. Whereas normalization with cel-miR-39 was adequate to reveal a trend toward lower let-7g levels in the PAH discovery cohort, this external control was ineffective in the larger validation cohort of subjects. However, lower let-7g levels were evident in PAH patients after normalization with the miR142-3p/miR-320a pair of internal reference controls. The quantification of extracellular miRNAs poses a unique technical Salubrinal challenge due to the relatively low levels at which they circulate in the blood, which precludes reliable assessment of RNA quantity and quality by traditional methods. The meaningful LY2090314 comparison of plasma miR levels among different subjects is further hampered by the lack of validated internal reference controls that circulate in blood at stable levels. Of note, the concept of plasma level stability addressed herein is distinct from chemical stability and analogous to the ‘expression’ stability of cellular housekeeping genes, which are characterized by their constitutive expression atrelatively constant levels in cells under both normal and pathophysiological conditions. Although thereisat present no consensus about the most effective strategy to normalize plasma miRNA levels, there is general agreement that more standardized approaches are important to improve reproducibility and facilitate the comparison of results between different investigative teams.