Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR

Brief Bioinform. 2016 Mar;17(2):204-12. doi: 10.1093/bib/bbv056. Epub 2015 Aug 3.


The high-throughput analysis of microRNAs (miRNAs) circulating within the blood of healthy and diseased individuals is an active area of biomarker research. Whereas quantitative real-time reverse transcription polymerase chain reaction (qPCR)-based methods are widely used, it is yet unresolved how the data should be normalized. Here, we show that a combination of different algorithms results in the identification of candidate reference miRNAs that can be exploited as normalizers, in both discovery and validation phases. Using the methodology considered here, we identify normalizers that are able to reduce nonbiological variation in the data and we present several case studies, to illustrate the relevance in the context of physiological or pathological scenarios. In conclusion, the discovery of stable reference miRNAs from high-throughput studies allows appropriate normalization of focused qPCR assays.

Keywords: Normfinder; circulating miRNA; geNorm; normalization; qPCR; reference genes.

MeSH terms

  • Algorithms*
  • Biomarkers / blood
  • Gene Expression Profiling / methods*
  • Gene Expression Profiling / standards
  • High-Throughput Nucleotide Sequencing / methods*
  • High-Throughput Nucleotide Sequencing / standards
  • Humans
  • MicroRNAs / blood*
  • MicroRNAs / genetics*
  • MicroRNAs / standards
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards
  • Reference Values
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Biomarkers
  • MicroRNAs