Sex and age differences in the expression of liver microRNAs during the life span of F344 rats

214 miRNAs differentially expressed by age and/or sex. Each miRNA is shown as differentially expressed by age, sex, or both age and sex, along with the k-means cluster number from Fig. 2 and chromosome mapping position. (XLSX 20 kb

Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements

BACKGROUND: Environmental ozone can rapidly degrade cyanine 5 (Cy5), a fluorescent dye commonly used in microarray gene expression studies. Cyanine 3 (Cy3) is much less affected by atmospheric ozone. Degradation of the Cy5 signal relative to the Cy3 signal in 2-color microarrays will adversely reduce the Cy5/Cy3 ratio resulting in unreliable microarray data. RESULTS: Ozone in central Arkansas typically ranges between ~22 ppb to ~46 ppb and can be as high as 60–100 ppb depending upon season, meteorological conditions, and time of day. These levels of ozone are common in many areas of the country during the summer. A carbon filter was installed in the laboratory air handling system to reduce ozone levels in the microarray laboratory. In addition, the airflow was balanced to prevent non-filtered air from entering the laboratory. These modifications reduced the ozone within the microarray laboratory to ~2–4 ppb. Data presented here document reductions in Cy5 signal on both in-house produced microarrays and commercial microarrays as a result of exposure to unfiltered air. Comparisons of identically hybridized microarrays exposed to either carbon-filtered or unfiltered air demonstrated the protective effect of carbon-filtration on microarray data as indicated by Cy5 and Cy3 intensities. LOWESS normalization of the data was not able to completely overcome the effect of ozone-induced reduction of Cy5 signal. Experiments were also conducted to examine the effects of high humidity on microarray quality. Modest, but significant, increases in Cy5 and Cy3 signal intensities were observed after 2 or 4 hours at 98–99% humidity compared to 42% humidity. CONCLUSION: Simple installation of carbon filters in the laboratory air handling system resulted in low and consistent ozone levels. This allowed the accurate determination of gene expression by microarray using Cy5 and Cy3 fluorescent dyes

Improvement in the Reproducibility and Accuracy of DNA Microarray Quantification by Optimizing Hybridization Conditions

BACKGROUND: DNA microarrays, which have been increasingly used to monitor mRNA transcripts at a global level, can provide detailed insight into cellular processes involved in response to drugs and toxins. This is leading to new understandings of signaling networks that operate in the cell, and the molecular basis of diseases. Custom printed oligonucleotide arrays have proven to be an effective way to facilitate the applications of DNA microarray technology. A successful microarray experiment, however, involves many steps: well-designed oligonucleotide probes, printing, RNA extraction and labeling, hybridization, and imaging. Optimization is essential to generate reliable microarray data. RESULTS: Hybridization and washing steps are crucial for a successful microarray experiment. By following the hybridization and washing conditions recommended by an oligonucleotide provider, it was found that the expression ratios were compressed greater than expected and data analysis revealed a high degree of non-specific binding. A series of experiments was conducted using rat mixed tissue RNA reference material (MTRRM) and other RNA samples to optimize the hybridization and washing conditions. The optimized hybridization and washing conditions greatly reduced the non-specific binding and improved the accuracy of spot intensity measurements. CONCLUSION: The results from the optimized hybridization and washing conditions greatly improved the reproducibility and accuracy of expression ratios. These experiments also suggested the importance of probe designs using better bioinformatics approaches and the need for common reference RNA samples for platform performance evaluation in order to fulfill the potential of DNA microarray technology

Un corps sans sexe ? Un procès en nullité de mariage et un verdict confondants dans la France du XIXe siècle

International audienceL e 29 avril 1869, se déroule au tribunal civil d'Alès le premier acte d'un procès qui connaît plusieurs rebondis-sements jusqu'en 1873 et un retentissement spectaculaire. Il n'est pas jusqu'au Petit Journal, journal populaire qui tire à plus de 300 000 exemplaires, qui y dépêche un correspondant particulier. Loin d'un crime sordide susceptible de doper les ventes du journal, il s'agit ici d'un contentieux relevant du privé. Plus encore, de l'intimité viscérale de chaque individu puisqu'un mari demande la nullité de son mariage au motif que sa femme, dépourvue des « organes naturels constitutifs de son sexe », n'en serait pas une (Tribunal civil d'Alès, 1869). Et voilà les magistrats, ici comme dans au moins vingt-quatre autres affaires du même type au XIX e siècle, face aux experts médicaux les plus fameux pour confondre le sexe suspect, composant avec le biologique quand le code civil de 1804 en avait été expurgé parles législateurs : une alliance de circons-tance qui va aboutir à un verdict pour le moins étonnant (G. Houbre, 2015). Antoine Darbousse et Justine Jumas se marient le 20 décembre 1866, à Alès, dans le Gard. Les époux appartiennent à deux vieilles familles de la bourgeoise protestante cévenole et se connaissent fort bien depuis l'enfance. Le mari, âgé de vingt-trois ans, s'illustre déjà dans la sériciculture, activité traditionnelle de la région. Justine Jumas, vingt-cinq ans, sans 1. Cet article puise une partie de son argumen-taire dans deux précédentes publications en 2012 et 2015

Analysis of gene expression changes in relation to toxicity and tumorigenesis in the livers of Big Blue transgenic rats fed comfrey (<it>Symphytum officinale</it>)

Abstract Background Comfrey is consumed by humans as a vegetable and a tea, and has been used as an herbal medicine for more than 2000 years. Comfrey, however, is hepatotoxic in livestock and humans and carcinogenic in experimental animals. Our previous study suggested that comfrey induces liver tumors by a genotoxic mechanism and that the pyrrolizidine alkaloids in the plant are responsible for mutation induction and tumor initiation in rat liver. Results In this study, we identified comfrey-induced gene expression profile in the livers of rats. Groups of 6 male transgenic Big Blue rats were fed a basal diet and a diet containing 8% comfrey roots, a dose that resulted in liver tumors in a previous carcinogenicity bioassay. The animals were treated for 12 weeks and sacrificed one day after the final treatment. We used a rat microarray containing 26,857 genes to perform genome-wide gene expression studies. Dietary comfrey resulted in marked changes in liver gene expression, as well as in significant decreases in the body weight and increases in liver mutant frequency. When a two-fold cutoff value and a P-value less than 0.01 were selected, 2,726 genes were identified as differentially expressed in comfrey-fed rats compared to control animals. Among these genes, there were 1,617 genes associated by Ingenuity Pathway Analysis with particular functions, and the differentially expressed genes in comfrey-fed rat livers were involved in metabolism, injury of endothelial cells, and liver injury and abnormalities, including liver fibrosis and cancer development. Conclusion The gene expression profile provides us a better understanding of underlying mechanisms for comfrey-induced hepatic toxicity. Integration of gene expression changes with known pathological changes can be used to formulate a mechanistic scheme for comfrey-induced liver toxicity and tumorigenesis.</p

K-means cluster analysis of differentially expressed genes.

<p>Differentially expressed genes were clustered into 28-means clusters (JMP Genomics, SAS 9.2) as this is the lowest number of clusters which allows a minimum correlation coefficient of R = 0.6 between any one expression profile and its other cluster members. The x- and y-axes represent age in weeks and relative fold change, respectively. Cluster color is arbitrary. These data illustrate the various biological patterns of kidney expression which exist during pre-pubertal, early adult and aged rat life stages.</p

Pathway analysis of prominent cluster groups representing life cycle stages.

a<p>Top Networks, BioFunctions, Canonical Pathways or Tox Lists from Ingenuity Pathway Analysis that showed repeated presence in top 5 lists for each cluster group.</p

Individual examples of gene expression profiles.

<p>Microarray data (black squares) and qRTPCR (blue squares, where present) fold-changes for selected DEGs are plotted (n = 4 or 5 for microarray data, n = 6 for qRTPCR, error bars represent SEM). Averaged microarray expression levels (normalized fluorescence intensities) across all time points are reported for each gene and have been scaled to equal 1 in the graph. Pearson correlation coefficients (R) between microarray and qRTPCR data are provided. All data is displayed, however x-axis labels for 5 and 6 weeks have been omitted due to space limitations.</p

Differential life cycle expression of genes encoding qualified kidney biomarkers.

<p>Genes encoding qualified renal biomarkers kidney injury molecule 1 (Kim-1), clusterin (Clu) and trefoil factor 3 (Tff3) show life cycle expression differences of 141-, 5- and 106-fold in the kidney, respectively. Microarray data (black squares) and qRTPCR (blue squares) relative fold changes are plotted (n = 4 or 5 for microarray data, n = 6 for qRTPCR, error bars represent SEM). Averaged microarray expression levels (normalized fluorescence intensities) across all time points are reported for each gene and have been scaled to equal 1 in the graph. All data is displayed, however x-axis labels for 5 and 6 weeks have been omitted due to space limitations.</p