|
| Status |
Public on Sep 22, 2014 |
| Title |
Muscle_Caloric_Restriction_Replicate #2 |
| Sample type |
RNA |
| |
|
| Source name |
Muscle_Caloric_Restricted_Diet
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
gender: male
tissue: Muscle
treatment: Standard AIN-93G diet, 40% Caloric restriction (CR)
|
| Treatment protocol |
Diets were started at 28 weeks of age after randomization into three groups. Mice were fed either a standard AIN-93G diet (SD; carbohydrate:protein:fat ratio of 64:19:17 percent of kcal), or a SD supplemented with SRT2104 at a dose of 1.33 g drug per kg of chow, formulated to provide daily doses of approximately 100 mg drug per kg body weight to the mice, or the SD diet at a 40% caloric restriction.
|
| Growth protocol |
Male C57BL/6J mice were started on three different diets at 28 weeks of age after randomization into three groups. Mice were placed either into a standard diet group, a standard diet plus SRT2104 group or a 40% caloric restriction group. Mice were allowed to live either to the end of their lives or until they were sacrificed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 41 weeks of age, after 14 weeks on each diet, muscle and liver tissue was harvested and snap frozen. RNA was extracted from both tissues using 1.0mm glass beads in a Precellys 24 Tissue Homogenizer and Qiagen RNeasy Mini Kits for the liver tissue or the RNeasy Fibrous Tissue kit for the muscle tissue according to manufacturer's specifications. Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips.
|
| Label |
Streptavidin-Cy3 bound to biotin labeled cRNA.
|
| Label protocol |
Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
|
| |
|
| Hybridization protocol |
Standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Sentrix MouseRef-8 v2 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has ~24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
|
| Scan protocol |
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
|
| Description |
Muscle_Caloric_Restriction_Replicate #2
|
| Data processing |
Data was extracted using the Illumina GenomeStudio software(v1.6.0). Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std. Complete data including detection scores will be included in the supplemental file.
Detection_Pval = Detection Pvalue from Illumina GenomeStudio software, ver 1.6.0.
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| |
|
| Submission date |
Feb 22, 2014 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL6885 |
| Series (1) |
| GSE49000 |
SRT2104 extends survival of male mice on a standard diet and preserves bone and muscle mass |
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