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.

Cholesterol synthesis is a ubiquitous and housekeeping metabolic pathway that leads to cholesterol, an essential structural component of mammalian cell membranes, required for proper membrane permeability and fluidity. The last part of the pathway involves steroidal triterpenes with cholestane ring structures. It starts by conversion of acyclic squalene into lanosterol, the first sterol intermediate of the pathway, followed by production of 20 structurally very similar steroidal triterpene molecules in over 11 complex enzyme reactions. Due to the structural similarities of sterol intermediates and the broad substrate specificity of the enzymes involved (especially sterol-Δ²⁴-reductase; DHCR24) the exact sequence of the reactions between lanosterol and cholesterol remains undefined. This article reviews all hitherto known structures of post-squalene steroidal triterpenes of cholesterol synthesis, their biological roles and the enzymes responsible for their synthesis. Furthermore, it summarises kinetic parameters of enzymes (V_max and K_m) and sterol intermediate concentrations from various tissues. Due to the complexity of the post-squalene cholesterol synthesis pathway, future studies will require a comprehensive meta-analysis of the pathway to elucidate the exact reaction sequence in different tissues, physiological or disease conditions. A major reason for the standstill of detailed late cholesterol synthesis research was the lack of several steroidal triterpene standards. We aid to this efforts by summarizing commercial and laboratory standards, referring also to chemical syntheses of meiosis-activating sterols.

24S- and 27-hydroxycholesterol (24OHC and 27OHC) are potent regulators of different biochemical systems in vitro and are the major circulating oxysterols. A small fraction of these oxysterols has been reported to be sulphated but there are no detailed studies. We considered the possibility that sulphatation is a protective mechanism preventing accumulation of free oxysterols. Using an accurate assay we found the sulphated fraction of 24OHC and 27OHC in circulation of adults to be less than 15% of total. In two patients with a mutation in CYP7B1 and markedly increased levels of 27OHC the sulphated fraction was 8% and 10% respectively. Infants with severe neonatal cholestasis had however markedly increased sulphate fraction of the above oxysterols. In untreated mice the degree of sulphatation of 24OHC and 27OHC in serum varied between 0 and 16%. Similar degree of sulphatation was found in two mouse models with markedly increased levels of 27OHC and 24OHC respectively. Bile duct ligated mice had higher levels of oxysterols than sham-operated controls but the sulphate fraction was not increased. We conclude that a primary increase in the levels of the oxysterols due to increased synthesis or reduced metabolism in adults and mice does not induce increased sulphatation.