Abstract
BackgroundCircadian 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.ResultsIn 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.ConclusionsGenetic 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.
Highlights
Circadian rhythms have a profound effect on human health
Selection and characteristics of candidate reference genes Seven candidate reference genes (Ppib, Rplp0, Gapdh, Actb, Hmbs, Hprt1 and Rn18s) were selected [20,23] and their expression measured in 35 livers and 33 adrenals from the inbred strain (C57BL/6JOlaHsd), 51 livers and 34 adrenals from the wild type mixed strain (129Pas plus C57BL/6J) and 45 livers and 30 adrenals from the mixed strain with a targeted disruption of the Crem gene (Crem KO)
In this study we investigated the most reliable reference genes for normalization of circadian studies within or between mouse strains in livers and adrenal glands
Summary
Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Circadian control is required for healthy life, disruption of circadian cycle leads to pathologies such as cancer, obesity, lipid disorders and type 2-diabetes [4,5,6]. Some of these abnormalities were discovered in mouse models lacking core clock genes Clock and Bmal1 [7,8,9].
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