biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsNazim Arda Kelestranscription profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15809A chronic or intermittent caloric restriction protocol was applied to mice from 10 weeks of age up to 82 weeks of age. Blood samples were collected at weeks 10 (baseline), 49/50 (adult), and old (81/82). Total RNA was isolated, and circulating miRNA profiling was performed using the Affymetrix GeneChip 4.1 miRNA microarray platform. Bioinformatic analyses were performed to identify age- and diet-associated circulating miRNAs in a lifelong CCR- or ICR-applied tumor-free breast cancer mouse model.biostudies-arrayexpressSample Collection - At the designated time points, 10 (baseline), 49 or 50 (adult), and 81 or 82 (old), blood samples were collected by retro-orbital bleeding after overnight fasting, and then the mice were sacrificed. Animals were anesthetized using isoflurane and humanely euthanized by isoflurane overdose under observation to prevent unexpected suffering. Blood samples were stored in a -80°C freezer until further use.Hybridization - Following the ligation step, samples were denatured in the hybridization master mix at 95°C for 5 mins, then at 45°C for 5 mins. Denaturized RNA samples were randomly hybridized using the GeneChip miRNA 4.1 array for 20 hours at 48°C at the GeneAtlas Hybridization Station.Nucleic Acid Extraction - Total RNA was isolated from the whole blood samples using the Jena Bioscience Total RNA Purification Kit (Jena Bioscience, Jena, Germany. CAT No: PP210-S). following the manufacturer’s instructions. Briefly, samples were lysed with the lysis buffer, followed by RNA binding to the spin column, on-column DNase I digestion to remove genomic DNA, washing, and elution in RNase-free water. The total RNA concentrations and purity were determined with a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) by measuring the absorbance at 260/280 and 260/230 ratios. RNA integrity was further evaluated by 1% agarose gel electrophoresis. The total RNA samples were stored in the -80°C freezer until further use.Scaning - Array strips were washed at the Affymetrix GeneAtlas Fluidics Station and visualized on Affymetrix Imaging Solution. The results were retrieved for each sample as raw CEL files.Growth Protocol - A total of 32 tumor-free mice were enrolled in this study (n = 8 for baseline, n = 3 or 4 for dietary groups). All mice were fed ad libitum (AL) until week 10 and then randomized into the three different groups. The health status of the mice was checked by the veterinarians and technicians at least once a week. Mice were allowed free access to tap water and were housed individually under standard conditions: a room temperature of 21-24°C and a 12-hour light/dark cycle. All procedures were performed in accordance with the guidelines and with the approval of the Yeditepe University Animal Care and Use Committee. All procedures involving animals complied with the European Community Council Directive of 24 November 1986, and ethical approval was granted by the Yeditepe University Ethics Committee (No. 11/03/2019, Istanbul, Turkiye).Sample Treatment - The dietary groups were AL, chronic CR (CCR), and intermittent CR (ICR). All mice were fed a standard chow diet (Altromin TPF1414) purchased from Kobay AS (Ankara, Turkiye). The AL group had free access to food throughout the study. A total of 85% of the daily food consumption of the age-matched AL group was provided to the CCR group, resulting in a 15% CR compared to the AL group. The ICR group was provided with 40% of the daily consumption of the age-matched AL group for a week, resulting in 60% CR, followed by AL feeding for three weeks. This protocol was applied to ICR mice in a cyclic period until they were sacrificed at the designated time points: week 10 (baseline), 49 or 50 (adult), and 81 or 82 (old). The mice in the ICR group were then divided into two groups: the ones that were sacrificed at the end of the 60% restriction period, called ICR-restricted (ICRR), and the ones that were sacrificed at the end of three weeks of AL period, called ICR-re-fed (ICRRF), to identify the effects of short-term refeeding. Body weights, food consumption, and tumor incidences were recorded weekly.Labeling - A total of 160 ng RNA from each sample, which showed good quality (A260/A280 = 2.0-2.1 nm), was exposed to a poly(A) tailing step with the addition of poly(A) tailing master mix and poly(A) Polymerase, then incubated at 37°C for 15 min. Samples were then labeled using FlashTag Biotin HSR RNA Labelling Kit (Applied Biosystems, Cat No: 901911). Biotin HSR Ligation was performed with the addition of a ligation mix that contained biotin-labeled RNA and T4 DNA Ligase. Then, samples were incubated at 25°C for 30 min.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsNazim Arda KelesData Transformation - Retrieved raw microarray data (.CEL files), with a sample annotation file, were imported into RStudio with the R programming language (version 4.2.2) for further analyses. The miRNA annotation file for mouse (Mus musculus) miRNA was provided commercially from Applied Biosystems GeneChip 4.1 as the feature data “miRNA-4_1-st-v1.annotations.20160922.csv” file (Thermo Fisher Scientific, USA). The count matrix was constructed using the raw CEL files, miRNA annotation, and sample annotation after quality control and data normalization with the oligo R package. Principal component analysis (PCA) was performed to reduce dimensionality and visualize the overall variance in the dataset using the PCAtools R package. A design matrix was constructed using group-timepoint combinations to identify differentially expressed (DE) miRNAs across aging and dietary groups. These contrasts included intra-group comparisons across time points (e.g., Adult vs. Baseline, AL group) and inter-group comparisons at the same time point (e.g., CCR vs. AL, Adult). Prior to the differential expression analysis, the count matrix data were filtered for the lowly expressed miRNAs. If the mean RMA normalized value of the detected miRNA probe in any of the experimental groups was lower than 0.4, it was removed. The linear models for microarray and omics data (limma) R package were used to evaluate differential expression of miRNAs among the groups. ComplexHeatmap and EnhancedVolcano R packages were used to visualize the most variable genes across samples and DE miRNAs between the groups. The MultiMiR R package was used to identify experimentally validated targets of the significant miRNAs, and these targets were considered if they were found in at least two of the three databases: miRRecords, miRBase, and TarBase. To determine the enriched Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, clusterProfiler R package was used. To identify previously reported breast cancer-associated miRNAs, the MultimiR R package was used with the “drug.disease” parameter set to “BREAST CANCER”, querying the databases miR2Disaese and PhenomiR. To identify the interactions between shared miRNAs, target genes, and enriched pathways, the alluvial plot was generated using the ggalluvial package. To optimize the visualization of the alluvial plot, the total number of target genes was limited to 20 genes.falseCirculating microRNA expression data of the caloric restriction applied breast cancer mouse modelA chronic or intermittent caloric restriction protocol was applied to mice from 10 weeks of age up to 82 weeks of age. Blood samples were collected at weeks 10 (baseline), 49/50 (adult), and old (81/82). Total RNA was isolated, and circulating miRNA profiling was performed using the Affymetrix GeneChip 4.1 miRNA microarray platform. Bioinformatic analyses were performed to identify age- and diet-associated circulating miRNAs in a lifelong CCR- or ICR-applied tumor-free breast cancer mouse model.2025-11-15T00:00:00Z2026-05-27T15:30:27.78Z2025-10-21T15:43:05.546ZE-MTAB-15809EFO_0002768EFO_0002944EFO_0003814EFO_0003813EFO_0003789EFO_0005518EFO_0003816EFO_0003815EFO_0003969