Materials and methods Animals C57BL 6. NOD Aec1Aec2 and C57BL 6J mice were bred and maintained under specific pathogen free conditions within selleck chem DZNeP the mouse facility of the Department of Pathology with over sight by Animal Care Services at the University of Florida, Gainesville. The animals were maintained on a 12 hour light dark schedule and provided food and acidified water ad libi tum. Although SjS in humans is most common in post meno pausal women, male mice were used exclusively in the present study as we have not noticed differences in the salivary gland disease in male and female C57BL 6. NOD Aec1Aec2 mice. Mice were euthanized at 4, 8, 12, 16, or 20 weeks of age by cervical dislocation after deep anesthetization with isoflurane. There are no indications that this procedure affects physiolog ical function of the exocrine glands.

Inhibitors,Modulators,Libraries Both the breeding and use of these animals for the present studies were approved by the University of Florida Institutional Animal Care and Use Com mittee. Salivary glands were freshly excised from individual male mice at 4, 8, 12, 16, or 20 weeks of age, snap frozen in liquid nitrogen, and stored at 80 C until all glandular samples were obtained. With one lobe of each sali vary gland, comprised of a submandibular, sublingual, and parotid gland minus any salivary lymph nodes, all 25 samples of total RNA from the five age groups of C57BL 6. NOD Aec1Aec2 mice were isolated concurrently using the RNeasy Mini Kit in accordance with the protocol of the manufacturer. To account for any asynchrony of SjS like disease within C57BL 6.

NOD Aec1Aec2 male mice, the Inhibitors,Modulators,Libraries five mice in each age group were derived from at least two litters. Hybridizations were carried out with each of the 25 individual RNA samples using Affymetrix GeneChip Mouse Genome 430 2. 0 Arrays in accordance with the instructions of the manufacturer. Each GeneChip Inhibitors,Modulators,Libraries contains 45,000 probe sets that ana lyze the expression level of over 39,000 transcripts and vari ants from over 34,000 well characterized mouse genes. Microarray data have been deposited with Gene Expression Omnibus accession number. Differential gene expression analysis Microarray data were normalized using Inhibitors,Modulators,Libraries the guanine cytosine robust multi array average algorithm and analyzed using the LIMMA pack age from the R Development Core Team to perform differential expression analyses.

Inhibitors,Modulators,Libraries LIMMA takes into account the correlation between replicates and uses the empirical Bayes approach, which gives stable inference for a relatively small number of arrays. In this study, the fdr method to adjust the P values for multiple testing was used to control the false discovery rate. Since the data represent five equally spaced time points, multiple models were used to identify the temporal patterns of gene Palbociclib CDK expression. These included the linear fit, quadratic fit, cubic fit, and quartic fit regression models. B statistics were calculated for each gene. Genes exhibiting a B sta tistic of greater than 1.