title: Technical factors involved in the measurement of circulating microRNA biomarkers for the detection of colorectal neoplasia.
abstract: BACKGROUND
  Circulating miRNAs are emerging as promising blood-based biomarkers for colorectal and other human cancers; however, technical factors that confound the development of these assays remain poorly understood and present a clinical challenge. The aim of this study was to systematically evaluate the effects of factors that may interfere with the accurate measurement of circulating miRNAs for clinical purposes.
  
  
  METHODS
  Blood samples from 53 subjects, including routinely drawn serum samples, matched plasma from 30 subjects, and matched serum samples drawn before and after bowel preparation for colonoscopy from 29 subjects were collected. Additionally, 38 serum specimens stored in the clinical laboratory for seven days were used to test the stability of miRNAs. Hemolysis controls with serial dilutions of hemoglobin were prepared. RNA was extracted from serum, plasma or hemolyzed controls with spiked-in cel-miR-39, and levels of miR-21, miR-29a, miR-125b and miR-16 were examined by real-time RT-PCR. Hemolysis was measured by spectrophotometry.
  
  
  RESULTS
  The expression levels of miR-16 and the degree of hemolysis were significantly higher in plasma than in serum (P<0.0001). Measured miR-21, miR-29a, miR-125b and miR-16 expression increased with hemoglobin levels in hemolyzed controls. The degree of hemolysis in serum samples correlated significantly with the levels of miR-21 (P<0.0001), miR-29a (P = 0.0002), miR-125b (P<0.0001) and miR-16 (P<0.0001). All four miRNAs showed significantly lower levels in sera that had been stored at 4°C for seven days (P<0.0001). Levels of miR-21 (P<0.0001), miR-29a (P<0.0001) and miR-16 (P = 0.0003), and the degree of hemolysis (P = 0.0002) were significantly higher in sera drawn after vs. before bowel preparation.
  
  
  CONCLUSIONS
  The measured levels of miRNAs in serum and plasma from same patients varied in the presence of hemolysis, and since hemolysis and other factors affected miRNA expression, it is important to consider these confounders while developing miRNA-based diagnostic assays.
authors.affiliation: Gastrointestinal Cancer Research Laboratory, Baylor Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, United States of America.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamada
authors.affiliation: Gastrointestinal Cancer Research Laboratory, Baylor Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, United States of America.
authors.forename: Mary A
authors.initials: MA
authors.lastname: Cox
authors.affiliation: Department of Internal Medicine, Baylor University Medical Center, Dallas, TX, United States of America.
authors.forename: Kristin A
authors.initials: KA
authors.lastname: Gaffney
authors.affiliation: Department of Internal Medicine, Baylor University Medical Center, Dallas, TX, United States of America.
authors.forename: Amber
authors.initials: A
authors.lastname: Moreland
authors.affiliation: Gastrointestinal Cancer Research Laboratory, Baylor Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, United States of America.
authors.forename: C Richard
authors.initials: CR
authors.lastname: Boland
authors.affiliation: Gastrointestinal Cancer Research Laboratory, Baylor Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, United States of America.
authors.forename: Ajay
authors.initials: A
authors.lastname: Goel
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood*; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000097:blood*; D055088:Early Detection of Cancer / Q000379:methods / Q000592:standards*; D006801:Humans; D035683:MicroRNAs / Q000097:blood*; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; D035683:MicroRNAs