A systemic approach to screening high-throughput RT-qPCR data for a suitable set of reference circulating miRNAs

BMC Genomics. 2020 Jan 31;21(1):111. doi: 10.1186/s12864-020-6530-3.


Background: The consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. We proposed a new in silico approach of finding a suitable reference for human, circulating miRNAs and identified a new set of endogenous reference miRNA based on miRNA profiling experiments from Gene Expression Omnibus. We used 3 known normalization algorithms (NormFinder, BestKeeper, GeNorm) to calculate a new normalization score. We searched for a universal set of endogenous miRNAs and validated our findings on 2 new datasets using our approach.

Results: We discovered and validated a set of 13 miRNAs (miR-222, miR-92a, miR-27a, miR-17, miR-24, miR-320a, miR-25, miR-126, miR-19b, miR-199a-3p, miR-30b, miR-30c, miR-374a) that can be used to create a reliable reference combination of 3 miRNAs. We showed that on average the mean of 3 miRNAs (p = 0.0002) and 2 miRNAs (p = 0.0031) were a better reference than single miRNA. The arithmetic means of 3 miRNAs: miR-24, miR-222 and miR-27a was shown to be the most stable combination of 3 miRNAs in validation sets.

Conclusions: No single miRNA was suitable as a universal reference in serum miRNA qPCR profiling, but it was possible to designate a set of miRNAs, which consistently contributed to most stable combinations.

MeSH terms

  • Algorithms
  • Circulating MicroRNA / genetics*
  • Computational Biology / methods*
  • Computer Simulation
  • Databases, Genetic
  • Gene Expression Profiling / standards
  • Humans
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards


  • Circulating MicroRNA