title: Evaluating normalization approaches for the better identification of aberrant microRNAs associated with hepatocellular carcinoma.
abstract: OBJECTIVE
  Dysregulated microRNAs (miRNAs) have been identified in hepatocellular carcinoma (HCC), but only a small proportion have been confirmed. An appropriate normalizer is crucial to determining the accuracy and reliability of data from miRNA studies.
  
  
  METHODS
  Different normalization strategies were used to validate genome-wide miRNA profiles in HCC tumor and non-tumor tissues, and to determine the consistency and discrepancy of data on dysregulated miRNAs.
  
  
  RESULTS
  Two sets of stable miRNAs (miR-30c/miR-30b and miR-30c/miR-126) were identified in HCC tissues by geNorm and NormFinder tools, respectively. The mean of global miRNAs also showed good stability for ranking the top 1-2 miRNAs, but the stabilities of the manufacturer-recommended ncRNAs controls were poor. Four panels of miRNAs were significantly associated with HCC by separately using various normalizers, and 14 miRNAs were consistently identified by three normalization strategies. Although fewer miRNAs (17-26) were dysregulated in HCC using the global mean or the 2 stable miRNAs as normalizers, perfect clustering of tissues was also obtained with only 1 to 2 misclassifications, suggesting the efficiency of the miRNA panels. Using global mean as the normalizer, the authors identified 7 miRNAs, including 2 novel (miR-324-5p and miR-550) significantly upregulated in HCC that were omitted when using 3 endogenous controls as the normalizer.
  
  
  CONCLUSIONS
  An optimal normalization strategy to identify biologically important miRNAs in HCC tissue studies of miRNA may be the combination of global mean and 2 stable miRNAs. Selection of appropriate normalization strategies to adjust miRNAs levels is particularly important for epidemiological studies dealing with large data sets and covering multiple experimental batches.
authors.affiliation: Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University Medical Center, New York, NY 10032, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Shen
authors.affiliation: Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University Medical Center, New York, NY 10032, USA.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University Medical Center, New York, NY 10032, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Gurvich
authors.affiliation: Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY 10032, USA.
authors.forename: Helen
authors.initials: H
authors.lastname: Remotti
authors.affiliation: Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University Medical Center, New York, NY 10032, USA.
authors.forename: Regina M
authors.initials: RM
authors.lastname: Santella
keywords: MicroRNA; biomarkers; hepatocellular carcinoma; normalization; stability