Reference genes for normalization: a study of rat brain tissue

Synapse. 2008 Apr;62(4):302-9. doi: 10.1002/syn.20496.


Quantitative real-time polymerase chain reaction (qPCR) has become a widely used tool in the search for disease genes. When examining gene expression with qPCR in psychiatric diseases, endogenous reference gene(s) must be used for normalization. Traditionally, genes such as beta-actin (ActB), Gapd, and 18s rRNA, assumed to be stably expressed, have been used for normalization. However, it has become clear that expression of these genes is influenced by different experimental paradigms. Since stable gene expression of houskeeping genes (HKGs), therefore, cannot be expected, alternative strategies are warranted. With the overall aim to inspect the gene expression of three target genes, NMDAR1, SORT, and CREB, in rat hippocampus, we tested a panel of eight HKGs, 18s rRNA, ActB, CycA, Gapd, Hmbs, Hprt1, Rpl13A, and Ywhaz in order to select the most stably expressed gene, using the NormFinder and geNorm software applets. Additionally, we have applied four different normalization approaches for normalization of the three target genes. We found using the NormFinder software that Ywhaz is the most stably expressed gene among the eight tested HKGs. However, the results of the analysis of the target genes are highly dependent on the choice of normalization approach. Moreover, the number of HKGs, used for selection of the most stable HKG, also influences on the result of the gene expression analysis of target genes. These results underline the importance of choosing a proper normalization strategy when analyzing gene expression with qPCR. The strategy should be unbiased and carried out in every specific experimental setting.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport
  • Animals
  • Brain / physiology*
  • Cyclic AMP Response Element-Binding Protein / genetics
  • Gene Expression Profiling / methods
  • Gene Expression Profiling / standards*
  • Gene Expression*
  • Male
  • Membrane Glycoproteins / genetics
  • Nerve Tissue Proteins / genetics
  • Rats
  • Receptors, N-Methyl-D-Aspartate / genetics
  • Reference Standards
  • Reverse Transcriptase Polymerase Chain Reaction
  • Software


  • Adaptor Proteins, Vesicular Transport
  • Cyclic AMP Response Element-Binding Protein
  • Membrane Glycoproteins
  • NMDA receptor A1
  • Nerve Tissue Proteins
  • Receptors, N-Methyl-D-Aspartate
  • sortilin