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Status |
Public on Jun 04, 2015 |
Title |
Cerebellum_WT_NR-Replicate-2 |
Sample type |
RNA |
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Source name |
Cerebellum_WT_Nicotinamide riboside
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 WT 3 months old treatment: WT mice, 3 months old were given subcutaneous interscapular injections of Nicotinamide riboside, 500 mg /kg body weight/day for 14 consecutive days at 4:00 pm. gender: male tissue collection: On day 15, the mice were sacrificed and half of the cerebellum was harvested for purification of mitochondria, with the left half snap-frozen, homogenized, and aliquoted for western blotting, microarray, and detection of ATP and NAD+.
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Treatment protocol |
CX and WT (C57BL/6) mice of 3 months of age were given subcutaneous interscapular injections of 500 mg NR/kg body weight/day or the equivalent volume of saline for a consecutive of 14 days at 4:00 pm
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Growth protocol |
Mice carrying WT, or CX (Csa-/-/Xpa-/-) alleles in a C57BL/6 background were maintained under standard laboratory conditions at Harvard School of Public Health and allowed free access to water and control casein pelleted diet (Research Diets D12450B).
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Extracted molecule |
total RNA |
Extraction protocol |
On day 15, mice were sacrificed and half of a cerebellum was harvested for purification of mitochondria, with the left half snap-frozen, homogenized, and aliquoted for RNA isolation. Total RNA extraction was done using a TRIzol Plus RNA purification kit as per manufacturer’s protocol. Quality and quantity of the total RNA was tested using the Agilent 2100 Bio-Analyzer and RNA 6000 nano kits.
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Label |
Streptavidin-Cy3 bound to biotin labeled cRNA.
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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.
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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.
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Scan protocol |
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
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Description |
Cerebellum_WT_NR-Replicate-2
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Data processing |
Data was extracted using the Illumina BeadStudio software(v1.9.0). 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.
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Submission date |
Mar 01, 2014 |
Last update date |
Jun 22, 2020 |
Contact name |
Supriyo De |
Organization name |
NIA-IRP, NIH
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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 |
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Platform ID |
GPL6885 |
Series (2) |
GSE55485 |
Defective Mitophagy in XPA via PARP1 activation and NAD+/SIRT1-depletion: Implications for neurodegeneration (mouse) |
GSE55486 |
Defective Mitophagy in XPA via PARP1 activation and NAD+/SIRT1-depletion: Implications for neurodegeneration |
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