Investigation Title Transcription profiling of skeletal muscles from young, old, and old calorie restricted C57BL/6NHsd mice to investigate age-related changes in the transcriptional profile Comment[Submitted Name] Expression data from skeletal muscle of young, old and old calorie restricted mice Experimental Design development_or_differentiation_design co-expression_design Experimental Design Term Source REF The MGED Ontology The MGED Ontology Comment[ArrayExpressReleaseDate] 2007-11-13 Comment[SecondaryAccession] GSE6323 Comment[AEMIAMESCORE] 5 Comment[SecondaryAccession] GDS2612 Comment[ArrayExpressAccession] E-GEOD-6323 Comment[MAGETAB TimeStamp_Version] 2010-08-06 20:11:27 Last Changed Rev: 13058 Comment[AEExperimentType] transcription profiling by array Experimental Factor Name diet age Experimental Factor Type diet age Person Last Name Edwards Person First Name Michael George Person Mid Initials Person Email medward71@yahoo.com Person Phone Person Fax Person Address Tomas Prolla, Genetics, University of Wisconsin, 425 Henry Mall, Madison, 53706, USA Person Affiliation University of Wisconsin Person Roles submitter Public Release Date 2007-11-13 PubMed ID 17381838 Publication DOI 10.1186/1471-2164-8-80 Publication Author List Michael G Edwards, Rozalyn M Anderson, Ming Yuan, Christina M Kendziorski, Richard Weindruch, Tomas A Prolla Publication Title Gene expression profiling of aging reveals activation of a p53-mediated transcriptional program. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description We investigated age-related changes in the transcriptional profile of skeletal muscle in 5 month old (young) and 25 month old (old) C57BL/6NHsd mice using high density oligonucleotide arrays (22,690 transcripts probed). We identified 712 transcripts that are differentially expressed in young (5 month old) and old (25-month old) mouse skeletal muscle. Caloric restriction (CR) completely or partially reversed 87% of the changes in expression. Examination of individual genes revealed a transcriptional profile indicative of increased p53 activity in the older muscle. To determine whether the increase in p53 activity is associated with transcriptional activation of apoptotic targets, we performed RT-PCR on four well known mediators of p53-induced apoptosis: puma, noxa, tnfrsf10b and bok. Expression levels for these proapoptotic genes increased significantly with age (P<0.05), while CR significantly lowered expression levels for these genes as compared to control fed old mice (P<0.05). Age-related induction of p53-related genes was observed in multiple tissues, but was not observed in SOD2+/- and GPX4+/- mice, suggesting that oxidative stress does not mediate the observed age-related increase in expression. Western blot analysis confirmed that protein levels for both p21 and GADD45a, two established transcriptional targets of p53, were higher in the older muscle tissue. These observations support a role for p53-mediated apoptotic activity in mammalian aging. Experiment Overall Design: Animals were killed by cervical dislocation. Five different animals were used for each experimental group. All collected gastrocnemius tissue was then dissected, placed in a microcentrifuge tube, flash-frozen in liquid nitrogen, and stored at -80°C until processed for microarrays. Protocol Name P-G6323-6 P-G6323-1 P-G6323-2 P-G6323-3 P-G6323-4 Protocol Type growth protocol treatment protocol nucleic acid extraction protocol nucleic acid labeling protocol nucleic acid hybridization to array protocol Protocol Description Male C57BL/6NHsd mice were purchased from Harlan Sprague-Dawley at 6-7 weeks of age and individually housed for the remainder of their lifespan. Mice were fed one of two life-long dietary regimens: normal diet (ND) and caloric restriction (CR). Mice on ND were provided acidic water ad libitum and fed 84 kcal/wk which is ~15% less than the average ad libitum diet. C57BL/6NHsd mice on this dietary regiment typically have an average lifespan of 30 mo. in our colony. Mice on CR were fed a diet that resulted in a 26% difference in caloric intake to the ND animals. The diet fed to CR mice was enriched in protein, vitamins and minerals such that CR and ND mice were fed nearly identical amounts of these components. Trizol extraction of total RNA was performed according to the manufacturer's instructions and polyadenylate [poly(A)+] RNA was purified from the total RNA with oligo-dT-linked Oligotex resin (Qiagen, Valencia, CA). One microgram of poly(A)+ RNA was converted into double-stranded cDNA (ds-cDNA) by using the SuperScript Choice System (Life Technologies) with an oligo-dT primer containing a T7 RNA polymerase promoter (Genset, La Jolla, CA). The ds-cDNA was extracted with phenol-chloroform-isoamyl alcohol and precipitated with pellet paint coprecipitant (Novagen, Madison, WI). Biotin-labeled RNA was synthesized in vitro using a high-yield RNA transcript labeling kit (BioArray; Enzo, Farmingdale, NY). The biotin-labeled antisense cRNA was purified using the RNeasy affinity column (Qiagen) and fragmented randomly. The hybridization cocktail (200 μl) containing 10 μg of fragmented cRNA was injected into the MOE430A array (Affymetrix, Santa Clara, CA). The GeneChip was placed in a 45°C oven at 60 rpm for 16 h. After hybridization, the GeneChips were washed and stained in a fluidic station (model 800101, Affymetrix) with signal amplification protocol using antibody. Protocol Term Source REF EFO EFO EFO EFO EFO SDRF File E-GEOD-6323.sdrf.txt Term Source Name The MGED Ontology ArrayExpress EFO Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ Term Source Version