snoU6 and 5S RNAs are not reliable miRNA reference genes in neuronal differentiation

Neuroscience. 2011 Dec 29;199:32-43. doi: 10.1016/j.neuroscience.2011.10.024. Epub 2011 Oct 20.


Accurate profiling of microRNAs (miRNAs) is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Quantitative real-time PCR (qPCR) has gained acceptance as a robust and reliable transcriptomic method to profile subtle changes in miRNA levels and requires reference genes for accurate normalization of gene expression. 5S and snoU6 RNAs are commonly used as reference genes in microRNA quantification. It is currently unknown if these small RNAs are stably expressed during neuronal differentiation. Panels of miRNAs have been suggested as alternative reference genes to 5S and snoU6 in various physiological contexts. To test the hypothesis that miRNAs may serve as stable references during neuronal differentiation, the expressions of eight miRNAs, 5S and snoU6 RNAs in five differentiating neuronal cell types were analyzed using qPCR. The stabilities of the expressions were evaluated using two complementary statistical approaches (geNorm and Normfinder). Expressions of 5S and snoU6 RNAs were stable under some but not all conditions of neuronal differentiation and thus are not suitable reference genes. In contrast, a combination of three miRNAs (miR-103, miR-106b and miR-26b) allowed accurate expression normalization across different models of neuronal differentiation.

MeSH terms

  • Animals
  • Cell Differentiation / genetics*
  • Cell Line
  • Gene Expression Profiling / standards*
  • Humans
  • Immunohistochemistry
  • Mice
  • MicroRNAs / analysis*
  • Neurons / cytology*
  • RNA Stability
  • RNA, Ribosomal, 5S / analysis*
  • RNA, Small Nuclear / analysis*
  • Rats
  • Real-Time Polymerase Chain Reaction / standards
  • Reference Standards
  • Reverse Transcriptase Polymerase Chain Reaction / standards


  • MicroRNAs
  • RNA, Ribosomal, 5S
  • RNA, Small Nuclear
  • U6 small nuclear RNA