title: Customized Internal Reference Controls for Improved Assessment of Circulating MicroRNAs in Disease.
abstract: BACKGROUND
  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. Here, we provide an approach to identify and validate circulating miRNA reference controls on a de novo basis, and demonstrate the advantages of these customized internal controls in different disease settings. Importantly, these internal controls overcome key limitations of external spike-in controls.
  
  
  METHODS
  Using a global RT-qPCR screen of 1066 miRNAs in plasma from pulmonary hypertension patients (PAH) and healthy subjects as a case example, we identified a large pool of initial candidate miRNAs that were systematically ranked according to their plasma level stability using a predefined algorithm. The performance of the top candidates was validated against multiple comparators, and in a second independent cohort of PAH and control subjects. The broader utility of this approach was demonstrated in a completely different disease setting with 372 miRNAs screened in plasma from septic shock patients and healthy controls.
  
  
  RESULTS
  Normalization of data with specific internal reference controls significantly reduced the overall variation in circulating miRNA levels between subjects (relative to raw data), provided a more balanced distribution of up- and down-regulated miRNAs, replicated the results obtained by the benchmark geometric averaging of all detected miRNAs, and outperformed the commonly used external spike-in strategy.
  
  
  CONCLUSIONS
  We demonstrate the feasibility of identifying circulating reference controls that can reduce extraneous technical variations, and improve the assessment of disease-related changes in plasma miRNA levels. This study provides a novel conceptual framework that addresses a critical and previously unmet need if circulating miRNAs are to advance as reliable diagnostic tools in medicine.
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute. Ottawa, Ontario, Canada.
authors.forename: Kenny
authors.initials: K
authors.lastname: Schlosser
authors.affiliation: Clinical Epidemiology Program, Ottawa Hospital Research Institute. Ottawa, Ontario, Canada.
authors.forename: Lauralyn A
authors.initials: LA
authors.lastname: McIntyre
authors.affiliation: Aab Cardiovascular Research Institute, University of Rochester, Rochester, New York, United States of America; Division of Pulmonary & Critical Care Medicine, University of Rochester, Rochester, New York, United States of America.
authors.forename: R James
authors.initials: RJ
authors.lastname: White
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute. Ottawa, Ontario, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Stewart
mesh_terms: D006976:Hypertension, Pulmonary / Q000097:blood* / Q000235:genetics; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012772:Shock, Septic / Q000097:blood* / Q000235:genetics
chemical_list: D035683:MicroRNAs