Customized internal reference controls for improved assessment of circulating microRNAs in disease

Altered levels of circulating extracellular miRNA in plasma and serum have shown promise as non-invasive biomarkers of disease. However, unlike the assessment of cellular miRNA levels for which there are accepted housekeeping genes, analogous reference controls for normalization of circulating miRNA are lacking. The objective of this study was to devise an approach to both identify and validate circulating miRNA reference controls on a de novo basis, and evaluate the relative performance of these customized internal reference controls versus other strategies. As a case example, 1066 different miRNAs were screened in plasma from pulmonary arterial hypertension patients and control subjects to identify an initial pool of miRNA candidates. PCR array profiling was performed on 4 treatment-naïve idiopathic pulmonary arterial hypertension (PAH) patients, 3 healthy control subjects and 1 non-PAH control subject