|
| Status |
Public on May 25, 2014 |
| Title |
Liver CREMKO 12h batch 5 [LiKOh12.36] |
| Sample type |
RNA |
| |
|
| Source name |
Whole liver RNA from Crem-/- mouse #18 at 12h
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Liver
strain: 129Pas x C57Bl/J
genotype/variation: CREMKO
animal: 18
time: 12
batch: 5
|
| Treatment protocol |
Mice were not treated.
|
| Growth protocol |
WT and Crem-/- animals of the mixed strain (129Pas x C57Bl/J ) [Nantel F, Monaco L, Foulkes NS, Masquilier D, LeMeur M, Henriksén K, Dierich A, Parvinen M, Sassone-Corsi P: Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice. Nature 1996, 380:159-162] were maintained in a temperature and humidity controlled room under a 12:12 h light:dark cycle (light on at 7:00 am, light off at 7:00 pm) with free access to food (Harland Tekland 2916) and water.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
8 weeks old mice were sacrificed with cervical dislocation at 7 am (0h circadian time) or 7 pm (12h circadian time) and tissue samples from liver, were excised, snap frozen in liquid nitrogen and stored at -80°C for subsequent analysis. Samples were first pulverized and then homogenized in 1000 μl of TRI Reagent (Sigma) and total RNA was isolated according to the manufacturer’s instructions. RNA quantity and quality were assessed with NanoDrop and Agilent 2100 Bioanalyzer instruments, all samples isolated fulfilled the minimum requirements (OD260/280 and OD230/280 > 1.8) for microarray hybridization.
|
| Label |
biotin
|
| Label protocol |
Total RNA was used to synthesize double-stranded cDNA with random hexamer primers tagged with a T7 promoter sequence. The double-stranded cDNA was subsequently used as a template and amplified by T7 RNA polymerase producing cRNA. In the second cycle of cDNA synthesis, random hexamers were used to prime reverse transcription of the cRNA and dUTP was incorporated to produce single-stranded DNA. This single-stranded DNA sample were then treated with a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) that specifically recognized the unnatural dUTP residues and broke the DNA strand. DNA was labeled by terminal eoxynucleotidyl transferase (TdT) with the Affymetrix proprietary DNA Labeling Reagent that is covalently linked to biotin.
|
| |
|
| Hybridization protocol |
100 ng of total RNA was used for hybridization of Affymetrix Gene Chip Mouse 1.0 ST Arrays according to manufacturer’s instructions (Genechip® Whole Transcript (wt) Sense Ttarget Labeling Assay Manual – P/N 701880 Rev 4). Labled samples were injected into Affymetrix arrays and hybridized at 45°C and 60 rpm for 16 h in an Affymetrix Hybridization Oven. Arrays were stained and washed in the Affymetrix GeneChip Fluidic Station 450.
|
| Scan protocol |
Arrays were scanned using Affymetrix GeneChip Scanner 3000 7G.
|
| Description |
Gene expression data from liver Crem-/- sample taken at 12h
|
| Data processing |
GeneChip data was processed and analyzed using R/Bioconductor packages. Data were summarized (w.r.t. core meta-probeset annotations and AFFX control probes, probes grouped to the level of transcripts) and quantile-normalized (background computed from antigenomic probes) using RMA algorithm from XPS package.
probe group file: MoGene-1_0-st-v1.r4.pgf
meta-probeset file: MoGene-1_0-st-v1.r4.mps
|
| |
|
| Submission date |
May 27, 2011 |
| Last update date |
May 25, 2014 |
| Contact name |
Peter Juvan |
| Phone |
+386 1 543 7595
|
| Organization name |
Faculty of Medicine, University of Ljubljana
|
| Department |
Institute of Biochemistry
|
| Lab |
Center for Functional Genomics and Bio-Chips
|
| Street address |
Zaloska 4
|
| City |
Ljubljana |
| ZIP/Postal code |
SI-1000 |
| Country |
Slovenia |
| |
|
| Platform ID |
GPL6246 |
| Series (2) |
| GSE29594 |
The effect of Crem absence on gene expression in mouse liver. |
| GSE29595 |
The effect of Crem absence on gene expression in mouse |
|
