Project description:In this study we utilized a genome-wide approach to analyze circadian patterns of gene expression in mouse lung lungs, both in the basal state and in the setting of systemic inflammation caused by endotoxemia. We chose the lung because it represents a primary portal for systemic infection and organ failure in critically ill patients, and because the lung exhibits strong physiological circadian rhythms in health and in diseases such as asthma. The gene expression the data presented here was correlated to histological observations and metabolite measurements derived from the same biological samples, in order to obtain a broad picture of how inflammation impacts circadian rhythms in mouse lung. To examine circadian regulation in mouse lung we performed 2 independent time-series experiments (Microarray Experiments #1 and #2), in which groups of mice were euthanized at 4 hour intervals for 48 hours. The purpose of Microarray Experiment #1 was to control for environmental influences (light and nutrition) on the detection of circadian rhythms in gene expression. In Microarray Experiment #1, 72 mice were segregated equally into 2 equal groups the day prior to the experiment at the beginning of the dark phase (Circadian Time 12 (CT12) or 7:00 PM local time). In one group (samples labeled with the suffix “MR”) the mice were kept under standard lighting and nutritional conditions (LD 12:12). In the second group (samples labeled with the suffix “DD”), mice were kept in constant darkness and food pellets were removed from their cages at the start of sample acquisition (DD 12:12+STV). Sample acquisition commenced at CT4 (or 11:00 AM local time). For each group 3 animals were sacrificed per time point (12 time points total). For mRNA isolation lung tissue was immersed in RNAlater (Qiagen) and total RNA was then extracted using the RNeasy Mini Kit (Qiagen). RNA quality (RIN>=7) was confirmed using an Agilent 2100 Bioanalyzer. RNA from all biological samples was labeled at once using the Ambion TotalPrep-96 RNA Amplification Kit. The samples were then blinded, randomized to chip position and hybridized to the Illumina MouseRef-8 v2.0 Expression BeadChips. For Microarray Experiment #1 RNA labeling and microarray hybridization were conducted at the Partners Center for Personalized Genetic Medicine. All biological samples were represented by a single technical replicate on the microarray.

Project description:The goal of the experiment was to perform a large scale study of circadian regulation of gene expression in maize. To identify maize genes with expression regulated by the circadian clock, transcript levels in the aerial tissues of young maize seedlings were determined by transcriptional profiling with the Affymetrix GeneChip Maize Genome Array. Maize inbred B73 seedlings were grown inside Conviron growth chamber. B73 seedlings were grown for 7 days under 12 h light:12 h dark (LD) photocycles, 26° C temperature and 70% humidity. At the 8th day, seedlings were transferred to continuous light (LL) and were allowed to entrain completely for 24 h prior to tissue harvest following which tissue was harvested every 4 hours under LL conditions for a period of 48h. Therefore, for the circadian LL time course 12 time points were collected as follows (also defined as factors in the treatment section): ZT0 - 8:00 am/ subjective dawn/ Day1 ZT4 - 12:00 pm/ subjective mid-day/ Day1 ZT8 - 4:00 pm/ subjective late-day/ Day1 ZT12 - 8:00 pm/ subjective dusk/ Day1 ZT16 - 12:00 am/ subjective mid-night/ Day1 ZT20 - 4:00 am/ subjective pre-dawn/ Day1 ZT24- 8:00 am/ subjective dawn/ Day2 ZT28 - 12:00 pm/ subjective mid-day/ Day2 ZT32 - 4:00 pm/ subjective late-day/ Day2 ZT36 - 8:00 pm/ subjective dusk/ Day2 ZT40 - 12:00 am/ subjective mid-night/ Day2 ZT44 - 4:00 am/ subjective pre-dawn/ Day2 Tissue comprised of aerial portion of the seedlings (corresponding to tissue from the prop roots and up) for RNA isolation. Total RNA was isolated from the entire aerial portion of 7 day-old seedlings (corresponding to tissue from the prop roots and up) by Trizol extraction followed by Qiagen RNeasy columns and treatment with RNase-free DNase I (Qiagen; qiagen.com). RNA was isolated from 3 independent biological replicates was pooled. cRNA was generated from pooled total RNA from 3 biological replicates with the GeneChip One-Cycle Target Labeling kit according to the manufacturer’s recommendations (Affymetrix, affymetrix.com). The University of California, Berkeley Functional Genomics Laboratory hybridized samples to Affymetrix GeneChip Maize Genome Arrays and scanned the washed arrays as suggested by manufacturer. Probe sets called “Not Present” or “Marginal” on one or more microarrays were removed from the downstream analysis, as is common practice with circadian studies. Raw hybridization intensities were normalized across all twelve arrays using RMA express in Perfect Match mode. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Frank G. Harmon. The equivalent experiment is ZM28 at PLEXdb.]

Project description:The goal of the experiment was to perform a large scale study of circadian regulation of gene expression in maize. To identify maize genes with expression regulated by the circadian clock, transcript levels in the aerial tissues of young maize seedlings were determined by transcriptional profiling with the Affymetrix GeneChip Maize Genome Array. Maize inbred B73 seedlings were grown inside Conviron growth chamber. B73 seedlings were grown for 7 days under 12 h light:12 h dark (LD) photocycles, 26° C temperature and 70% humidity. At the 8th day, seedlings were transferred to continuous light (LL) and were allowed to entrain completely for 24 h prior to tissue harvest following which tissue was harvested every 4 hours under LL conditions for a period of 48h. Therefore, for the circadian LL time course 12 time points were collected as follows (also defined as factors in the treatment section): ZT0 - 8:00 am/ subjective dawn/ Day1 ZT4 - 12:00 pm/ subjective mid-day/ Day1 ZT8 - 4:00 pm/ subjective late-day/ Day1 ZT12 - 8:00 pm/ subjective dusk/ Day1 ZT16 - 12:00 am/ subjective mid-night/ Day1 ZT20 - 4:00 am/ subjective pre-dawn/ Day1 ZT24- 8:00 am/ subjective dawn/ Day2 ZT28 - 12:00 pm/ subjective mid-day/ Day2 ZT32 - 4:00 pm/ subjective late-day/ Day2 ZT36 - 8:00 pm/ subjective dusk/ Day2 ZT40 - 12:00 am/ subjective mid-night/ Day2 ZT44 - 4:00 am/ subjective pre-dawn/ Day2 Tissue comprised of aerial portion of the seedlings (corresponding to tissue from the prop roots and up) for RNA isolation. Total RNA was isolated from the entire aerial portion of 7 day-old seedlings (corresponding to tissue from the prop roots and up) by Trizol extraction followed by Qiagen RNeasy columns and treatment with RNase-free DNase I (Qiagen; qiagen.com). RNA was isolated from 3 independent biological replicates was pooled. cRNA was generated from pooled total RNA from 3 biological replicates with the GeneChip One-Cycle Target Labeling kit according to the manufacturer’s recommendations (Affymetrix, affymetrix.com). The University of California, Berkeley Functional Genomics Laboratory hybridized samples to Affymetrix GeneChip Maize Genome Arrays and scanned the washed arrays as suggested by manufacturer. Probe sets called “Not Present” or “Marginal” on one or more microarrays were removed from the downstream analysis, as is common practice with circadian studies. Raw hybridization intensities were normalized across all twelve arrays using RMA express in Perfect Match mode. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Frank G. Harmon. The equivalent experiment is ZM28 at PLEXdb.] Continuous Light: ZT0(1-replications); Continuous Light: ZT4(1-replications); Continuous Light: ZT8(1-replications); Continuous Light: ZT12(1-replications); Continuous Light: ZT16(1-replications); Continuous Light: ZT20(1-replications); Continuous Light: ZT24(1-replications); Continuous Light: ZT28(1-replications); Continuous Light: ZT32(1-replications); Continuous Light: ZT36(1-replications); Continuous Light: ZT40(1-replications); Continuous Light: ZT44(1-replications)

Dataset Information

CIRCADIAN RHYTHM REPROGRAMMING DURING LUNG INFLAMMATION/ Microarray Experiment #2

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