title: Determination of reference genes for circadian studies in different tissues and mouse strains.
abstract: BACKGROUND
  Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration.
  
  
  RESULTS
  In the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons.
  
  
  CONCLUSIONS
  Genetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.
authors: Faculty of Medicine, Center for Functional Genomics and Bio-Chips, Institute of Biochemistry, University of Ljubljana, Zaloska 4, SI-1000 Ljubljana, Slovenia.
authors: Rok
authors: R
authors: Kosir
authors: Jure
authors: J
authors: Acimovic
authors: Marko
authors: M
authors: Golicnik
authors: Martina
authors: M
authors: Perse
authors: Gregor
authors: G
authors: Majdic
authors: Martina
authors: M
authors: Fink
authors: Damjana
authors: D
authors: Rozman
mesh_terms: D000818:Animals; D002940:Circadian Rhythm / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards