|
| Status |
Public on Apr 23, 2015 |
| Title |
Cortex ApoD-KO Old, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse cortex, 655 days old
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
gender: Male
tissue: Brain cortex
genotype: ApoD-KO
age: 655 days old
|
| Growth protocol |
Mice were maintained in positive pressure-ventilated racks at 25±1ºC with 12 h light/dark cycle, fed ad libitum with a standard rodent pellet diet (Global Diet 2014; Harlan Inc., Indianapolis, IN, USA), and allowed free access to filtered and UV-irradiated water.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mouse brains were quickly removed following decapitation after CO2 anesthesia. Hippocampal and cortical regions selected for mRNA expression studies were rapidly sliced in cold PBS under a stereomicroscope, and stored at -80ºC. RNA was extracted with QIAzolTM Lysis Reagent (Qiagen) followed by a second purification step with RNeasy miniKit (Qiagen) The purified RNA quality was assayed using the Agilent 2100 Bioanalyzer. Equimolar amounts of total RNA from 2-3 randomly selected mice for each genotype, brain region and age were used to make pools. Three of these biological replicates per condition were used for hybridization with oligonucleotide microarrays.
|
| Label |
biotin
|
| Label protocol |
cDNA was synthesized and purified from 5 micrograms of each RNA sample with the One Cycle cDNA synthesis (Affymetrix, Santa Clara, CA). The generation, labeling and purification of cRNA were performed using the IVT kit (Affymetrix, Santa Clara, CA). Probe synthesis and hybridizations were performed at the Genomics facility of the Centro de Investigacion del Cancer (Salamanca, Spain).
|
| |
|
| Hybridization protocol |
2.3 micrograms of the biotinylated and fragmented probes were hybridized to Affymetrix chips at 45ºC for 16 h, following the manufacturer's protocols.
|
| Scan protocol |
Following labeling and washes, the arrays were incubated with anti-biotin streptavidin-phycoeritrin antibody and were scanned with an Affymetrix GeneChip Scanner 7G.
|
| Data processing |
Analysis of differential gene expression was performed with FlexArray v1.6.1 program (Blazejczyk and others 2007), using the Affymetrix CEL files. Robust normalization using MAS 5.0 was performed to estimate a change p-value and its associated change call in gene expression for each probeset. Data pre-processing was carried out with the robust multiarray average algorithm (RMA) with background correction and normalization. Gene differential expression was evaluated with FlexArray by two sample comparisons with the cyberT-test, using a significance threshold of 1.2-fold change and a P value <0.05. False discovery rate (FDR) correction (5%) was performed using the Benjamini-Hochberg method. A Significance Analysis of Microarrays (SAM) algorithm using an unequal variance t-statistic was also employed to select genes robustly affected by age and/or genotype. Probe sets derived from non-annotated genes were not considered for the final discussion of differentially expressed genes.
|
| |
|
| Submission date |
Apr 22, 2015 |
| Last update date |
Apr 23, 2015 |
| Contact name |
Diego Sanchez |
| E-mail(s) |
[email protected]
|
| Phone |
+34 983184814
|
| Organization name |
Universidad de Valladolid
|
| Department |
IBGM
|
| Lab |
C5
|
| Street address |
C/ Sanz y Forés 3
|
| City |
Valladolid |
| State/province |
Valladolid |
| ZIP/Postal code |
47003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL19192 |
| Series (1) |
| GSE68169 |
Expression data from mouse brain |
|
