Determination of reference genes for circadian studies in different tissues and mouse strains

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>In the present study the expression of ten candidate reference genes (<it>Actb</it>, <it>Eif2a</it>, <it>Gapdh</it>, <it>Hmbs</it>, <it>Hprt1</it>, <it>Ppib</it>, <it>Rn18s</it>, <it>Rplp0</it>, <it>Tbcc </it>and <it>Utp6c</it>) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and <it>Crem </it>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 (<it>Actb</it>) 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.</p> <p>Conclusions</p> <p>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.</p

Cholesterol, Oxysterol, Triglyceride, and Coenzyme Q Homeostasis in ALS. Evidence against the Hypothesis That Elevated 27-Hydroxycholesterol Is a Pathogenic Factor

High plasma levels of cholesterol have been suggested to be neuroprotective for the degenerative disease amyotrophic lateral sclerosis (ALS) and to be associated with increased survival time. The gene encoding cholesterol 27-hydroxylase, CYP27A1, was recently identified as a susceptibility gene for sporadic ALS. A product of this enzyme is 27-hydroxycholesterol. We investigated plasma samples from 52 ALS patients and 40 control subjects (spouses) regarding cholesterol homeostasis, lipid profiles, and coenzyme Q. Eleven of the patients carried mutations in C9orf72 and seven in SOD1. Plasma levels of 27-hydroxycholesterol were significantly lower in male patients with ALS than in controls. It was not possible to link the reduced levels to any specific mutation, and there was no significant correlation between 27-hydroxycholesterol and survival. With normalization for diet using the spouses, a correlation was found between survival and total cholesterol, very low density lipoprotein cholesterol, low density lipoprotein cholesterol, and coenzyme Q. We conclude that cholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol and lipid profiles in plasma are of limited prognostic value in individual ALS patients

Cholesterol, Oxysterol, Triglyceride, and Coenzyme Q Homeostasis in ALS. Evidence against the Hypothesis That Elevated 27- Hydroxycholesterol Is a Pathogenic Factor

Abstract High plasma levels of cholesterol have been suggested to be neuroprotective for the degenerative disease amyotrophic lateral sclerosis (ALS) and to be associated with increased survival time. The gene encoding cholesterol 27-hydroxylase, CYP27A1, was recently identified as a susceptibility gene for sporadic ALS. A product of this enzyme is 27-hydroxycholesterol. We investigated plasma samples from 52 ALS patients and 40 control subjects (spouses) regarding cholesterol homeostasis, lipid profiles, and coenzyme Q. Eleven of the patients carried mutations in C9orf72 and seven in SOD1. Plasma levels of 27-hydroxycholesterol were significantly lower in male patients with ALS than in controls. It was not possible to link the reduced levels to any specific mutation, and there was no significant correlation between 27-hydroxycholesterol and survival. With normalization for diet using the spouses, a correlation was found between survival and total cholesterol, very low density lipoprotein cholesterol, low density lipoprotein cholesterol, and coenzyme Q. We conclude that cholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol and lipid profiles in plasma are of limited prognostic value in individual ALS patients

Multivariate analysis using PCA of the parameters in tables 1 and 2 (with BMI and age excluded) using two significant principal components based on eigenvalue criteria (>2).

<p>A. Females with ALS overlapped with female controls in the measured parameters. (t <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113619#pone.0113619-Andersen1" target="_blank">[1]</a>, 34% of variation vs. t <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113619#pone.0113619-Desport1" target="_blank">[2]</a>, 20% of variation; R2 = 0.67; Q2 = .25). B) The male ALS group was shifted from the male controls in the measured parameters. (t <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113619#pone.0113619-Andersen1" target="_blank">[1]</a>, 36% of variation vs. t <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113619#pone.0113619-Desport1" target="_blank">[2]</a>, 15% of variation; R2 = 0.51; Q2 = 0.22).</p

Female subjects Average values ± standard deviation for parameters measured for female controls and ALS patients, fold change (ALS/controls), and p-values for females.

<p>Female subjects Average values ± standard deviation for parameters measured for female controls and ALS patients, fold change (ALS/controls), and p-values for females.</p

Male subjects Average values ± standard deviation for parameters measured for male controls, ALS patients, fold change (ALS/controls), and p-values for males.

<p>Male subjects Average values ± standard deviation for parameters measured for male controls, ALS patients, fold change (ALS/controls), and p-values for males.</p

Correlation to survival Pearson's correlation for the parameters plotted against survival from time of sampling for all subjects and Spearman's correlation for the parameters plotted against survival from time of sampling for females and males.

<p>Correlation to survival Pearson's correlation for the parameters plotted against survival from time of sampling for all subjects and Spearman's correlation for the parameters plotted against survival from time of sampling for females and males.</p

OPLS model predicting survival from the time of sampling for diet-matched ALS cases (p = 0.003).

<p>A. Survival predicted by model plotted against time of survival from sampling. B. Long survival from sampling correlated with higher levels of LDL cholesterol, total cholesterol, coenzyme Q, and VLDL cholesterol.</p

Variability in the measured parameters in one female patient with sporadic pseudo bulbar paresis, the samples were collected over 11 months with one time-point in duplicate.

<p>Variability in the measured parameters in one female patient with sporadic pseudo bulbar paresis, the samples were collected over 11 months with one time-point in duplicate.</p