title: Analysis of circulating microRNA: preanalytical and analytical challenges.
abstract: BACKGROUND
  There is great interest in circulating microRNAs (miRNAs) as disease biomarkers. Translating promising miRNAs into validated clinical tests requires the characterization of many preanalytical and analytical parameters.
  
  
  METHODS
  miRNAs were extracted from serum and plasma samples of healthy volunteers, and miRNAs known to be present in serum and plasma (miR-15b, miR-16, miR-24, and miR-122) were amplified by reverse-transcription quantitative PCR. Stability and the effects of hemolysis were determined. Assay variation and its components, including the effect of adding control miRNA, were assessed by nested ANOVA.
  
  
  RESULTS
  miRNA concentrations were higher in plasma than in serum. Processing of plasma to remove subcellular/cellular components reduced miRNA concentrations to those of serum. The miRNAs analyzed were stable refrigerated or frozen for up to 72 h and were stable at room temperature for 24 h. Hemolysis increased the apparent concentration of 3 of the miRNAs. The total variability of replicate miRNA concentrations was <2.0-fold, with most of the variability attributable to the extraction process and interassay imprecision. Normalizing results to those of spiked exogenous control miRNAs did not improve this variability.
  
  
  CONCLUSIONS
  Detailed validation of the preanalytical steps affecting miRNA detection and quantification is critical when considering the use of individual miRNAs as clinical biomarkers. Unless these causes of imprecision are considered and mitigated, only miRNAs that are extremely up- or downregulated will be suitable as clinical biomarkers.
authors.affiliation: Department of Laboratory Medicine and Pathology, MayoClinic, Rochester, MN 55905, USA.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: McDonald
authors.forename: Dragana
authors.initials: D
authors.lastname: Milosevic
authors.forename: Honey V
authors.initials: HV
authors.lastname: Reddi
authors.forename: Stefan K
authors.initials: SK
authors.lastname: Grebe
authors.forename: Alicia
authors.initials: A
authors.lastname: Algeciras-Schimnich
mesh_terms: D005260:Female; D006461:Hemolysis; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000097:blood*; D020871:RNA Stability; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: C531907:MIRN122 microRNA, human; C505361:MIRN15 microRNA, human; C505362:MIRN16 microRNA, human; C543845:MIRN24 microRNA, human; D035683:MicroRNAs