|
| Status |
Public on Jul 30, 2014 |
| Title |
Sirt6_KO_retina_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse total retinal RNA from Sirt6 KO mouse
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129SvJ
age: 15 days old
genotype/variation: Sirt6 KO
tissue: retina
|
| Treatment protocol |
Retinas were homogenized in phenol-guanidine isothiocyanate (Invitrogen Life Technologies).
|
| Growth protocol |
Total retinal RNA from 15 day old Sirt6 wild-type (WT) and knock-out (KO) mice.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with chloroform and precipitated with isopropanol by centrifugation (12,000g) at 4°C. The RNA pellet was washed twice with 75% ethanol and resuspended in 30 μL of water treated with diethylpyrocarbonate. The concentration and quality of total RNA samples were assessed using a NANODROP 2000 spectrophotometer (Thermo Scientific Inc., Newark, DE). Only RNA samples with adequate purity ratios (A260/A280 ~ 1.9–2.1) were used for subsequent analyses. Total RNA was isolated using RNeasy Mini Kit (QIAGEN) and cDNA was generated using QuantiTect Reverse Transcription Kit (QIAGEN) according to manufacturer instructions.
|
| Label |
biotin
|
| Label protocol |
First strand cDNA synthesis was generated using a T7-linked oligo-dT primer, followed by second strand synthesis. An in vitro transcription reaction was performed to generate cRNA containing biotinylated UTP and CTP, which was subsequently chemically fragmented at 95°C for 35 minutes.
|
| |
|
| Hybridization protocol |
Ten micrograms of the fragmented, biotinylated cRNA was hybridized in MES buffer (2-[N-Morpholino]ethansulfonic acid) containing 0.5 mg/ml acetylated bovine serum albumin (Sigma) to Affymetrix MoGene-2_1-st arrays at 45°C for 16 hours.
|
| Scan protocol |
Scans were performed on Affymetrix scanners.
|
| Description |
whole retina mRNA from Sirt6 knock-out mice replicate 6/6
Sirt6_KO_6_6
|
| Data processing |
Raw expression values were normalized using Robust Multiarray Averaging (RMA).
|
| |
|
| Submission date |
Apr 07, 2014 |
| Last update date |
Jul 31, 2014 |
| Contact name |
Kenneth N Ross |
| E-mail(s) |
Kenneth.Ross@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Pediatric Oncology
|
| Street address |
450 Brookline Ave., Rm M640
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE56563 |
SIRT6 regulates glucose metabolism and glutamatergic synapse in the mouse retina |
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