Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Biological Aging and Circadian Mechanisms in Murine Brown Adipose Tissue, Inguinal White Adipose Tissue, and Liver (Nov 2009 dataset)

ABSTRACT: The circadian profile of the transcriptomes in murine tissues was compared between groups of young and old male mice. The oscillatory phase and absolute expression levels were evaluated as a function of biological age. Distinct differences in multiple pathways were apparent based on biological and circadian time in a tissue specific manner. A total of 18 young (5 month) and old (24 month) C57BL/6 mice were acclimated to a set 12 hr light dark cycle and fed ad lib on standard lab chow for 2 weeks in the PBRC Comparative Biology Core Facility. Five days prior to the study, the mice were converted to constant darkness (red light) regimen. On the day of the study, mice were euthanized by CO2 asphyxiation after ad lib feeding overnight in groups of 3 animals per young or old cohort at 4 hr intervals beginning at 7 AM and ending at 3 AM on a single day (July, 2009). The body weight of each animal was recorded prior to dissection of the following tissues: brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and liver. Individual tissues were weighed prior to freezing in liquid nitrogen. Samples were stored at -80oC until use.Total RNA was isolated from the BAT, eWAT, and liver tissues using TriReagent (MRC, Cincinnati OH) in accordance with the manufacturer’s recommendations. The Illumina TotalPrep RNA Amplification Kit (Applied Biosystems Inc., Foster City, CA, Catalog #AMIL1791) was used to create labeled cRNA from 750ng of input total RNA according to the manufacturer’s protocol. The labeled cRNA samples were then assessed for quality and quantity using a NanoDrop and an Agilent Bioanalyzer. The MouseWG-6 v2 Beadchip (Illumina Sentrix Beadchip Array #11278593) contains 45,200 transcripts and allows 6 samples to be interrogated in parallel. 1.5ug of each labeled cRNA was hybridized to each array according to the manufacturer's protocol. Experimental group samples were distributed randomly across all beadchips. After an 18 hour hybridization at 58°C, the beadchips were processed according to manufacturer’s protocol and scanned using an Illumina BeadArray Reader (Illumina, Inc., San Diego, CA).

ORGANISM(S): Mus musculus

SUBMITTER: Jeffrey Gimble

PROVIDER: E-GEOD-25323 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Biological aging alters circadian mechanisms in murine adipose tissue depots.

Sutton Gregory M GM Ptitsyn Andrey A AA Floyd Z Elizabeth ZE Yu Gang G Wu Xiying X Hamel Katie K Shah Forum S FS Centanni Armand A Eilertsen Kenneth K Kheterpal Indu I Newman Susan S Leonardi Claudia C Freitas Michael A MA Bunnell Bruce A BA Gimble Jeffrey M JM

Age (Dordrecht, Netherlands) 20120313 3

Biological aging alters the metabolism and volume of adipose tissue depots. Recent evidence suggests that circadian mechanisms play a role in promoting adipogenesis, obesity, and lipodystrophy. The current study compared cohorts of younger (5-9 months) and older (24-28 months) C57BL/6 mice as a function of biological age and circadian time. Advanced age significantly reduced the weight of the brown, epididymal, inguinal, and retroperitoneal adipose depots but not total body weight. The older mic ...[more]

PMID: 22411258

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Project description:The circadian profile of the transcriptomes in murine tissues was compared between groups of young and old male mice. The oscillatory phase and absolute expression levels were evaluated as a function of biological age. Distinct differences in multiple pathways were apparent based on biological and circadian time in a tissue specific manner. A total of 18 young (5 month) and old (24 month) C57BL/6 mice were acclimated to a set 12 hr light dark cycle and fed ad lib on standard lab chow for 2 weeks in the PBRC Comparative Biology Core Facility. Five days prior to the study, the mice were converted to constant darkness (red light) regimen. On the day of the study, mice were euthanized by CO2 asphyxiation after ad lib feeding overnight in groups of 3 animals per young or old cohort at 4 hr intervals beginning at 7 AM and ending at 3 AM on a single day (July, 2009). The body weight of each animal was recorded prior to dissection of the following tissues: brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and liver. Individual tissues were weighed prior to freezing in liquid nitrogen. Samples were stored at -80oC until use.Total RNA was isolated from the BAT, eWAT, and liver tissues using TriReagent (MRC, Cincinnati OH) in accordance with the manufacturer’s recommendations. The Illumina TotalPrep RNA Amplification Kit (Applied Biosystems Inc., Foster City, CA, Catalog #AMIL1791) was used to create labeled cRNA from 750ng of input total RNA according to the manufacturer’s protocol. The labeled cRNA samples were then assessed for quality and quantity using a NanoDrop and an Agilent Bioanalyzer. The MouseWG-6 v2 Beadchip (Illumina Sentrix Beadchip Array #11278593) contains 45,200 transcripts and allows 6 samples to be interrogated in parallel. 1.5ug of each labeled cRNA was hybridized to each array according to the manufacturer's protocol. Experimental group samples were distributed randomly across all beadchips. After an 18 hour hybridization at 58°C, the beadchips were processed according to manufacturer’s protocol and scanned using an Illumina BeadArray Reader (Illumina, Inc., San Diego, CA).

Project description:We have used commercially available oligonucleotide arrays to measure the relative expression levels of >10,000 genes and ESTs. Recent evidence suggested certain advantages of performing such expression experiments on two or more different platforms of oligonucleotide arrays. Here we have used two oligonucleotide platforms, MG U74Av2.0 arrays from Affymetrix and CodeLink UniSet1 from GE Healthcare, to assess whole brain gene expression patterns in naive C57BL/6 and DBA/2J mice. C57BL/6J (n = 5) and DBA/2J (n = 4) male mice, 25-30 g body weight, were obtained from Jackson Laboratories. Animals were group-housed and quickly sacrificed by CO2 exposure according to a protocol approved by the UCHSC IACUC. Brains were rapidly removed and stored at -80oC until use. Whole brain was homogenized in lysis buffer using a Polytron, and total RNA was isolated using the RNeasy Midi Kit (Qiagen) following the protocol supplied by the manufacturer. An additional clean-up of total RNA was carried out using the RNeasy Mini Kit (Qiagen). Using the protocol supplied by the manufacturer, double-stranded cDNA was synthesized from the total RNA (4 ug of total RNA from an individual mouse brain was used for each array) and was used to obtain biotin-labeled cRNA by an in vitro transcription reaction. Biotin-labeled cRNA was recovered using the RNeasy kit. Biotin-labeled cRNA from each mouse was then fragmented and hybridized with two separate (duplicate) CodeLink BioArrays. The average of the gene expression intensity values for each probe set from these technical replicates was used for data analysis. The BioArrays were subsequently stained for 30 min with Cy5-Streptavidin and washed prior to scanning. For MG U74Av2 arrays,double-stranded cDNA was synthesized from 5 ug of total RNA, using the protocol supplied by the manufacturer, and used to obtain biotin-labeled cRNA by an in vitro transcription reaction. Biotin-labeled cRNA was then fragmented and the quality of the fragmented cRNA was assessed using Test2 chips prior to hybridization with the Affymetrix arrays. Hybridization of the cRNA with the Affymetrix arrays was carried out according to the manufacturer's protocol. The arrays were stained with streptavidin-phycoerythrin conjugate and scanned by a GeneArray scanner. Keywords: other

Dataset Information

Biological Aging and Circadian Mechanisms in Murine Brown Adipose Tissue, Inguinal White Adipose Tissue, and Liver (Nov 2009 dataset)

Publications

Biological aging alters circadian mechanisms in murine adipose tissue depots.

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