|
| Status |
Public on Jan 08, 2010 |
| Title |
Prnp KO repl4 |
| Sample type |
RNA |
| |
|
| Source name |
Prnp knock out hippocampus
|
| Organism |
Mus musculus |
| Characteristics |
gender: adult male
age: 3-5 months old
weight: 25-35 g
status: loss of function of Prnp gene
strain: Prnp -/-
|
| Growth protocol |
The Prnp -/- mice used in this study were purchased from EMMA (Monterotondo, Italy). Originally described by Büeler et al. in 1992, these mice showed mixed background with 129/Ola and C57BL/6J. We back-crossed to C57BL/6J for at least 15 generations to obtain Prnp -/- and control littermates. PrPc overexpressing mice (Tg20) were purchased from EMMA (Monterotondo). These animals were generated by random insertion in Prnp -/- mice described by Marek et al (Fischer et al., 1996).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol followed by cleanup with the RNAsy Kit (Qiagen). RNA concentration was measured with a small volume spectrophotometer (Nanodrop). The quality of the RNA was analyzed using the Agilent Bioanalyzer 2100 and the RNA 6000 LabChip Kit (Agilent) with the Eukaryote Total RNA Nano Assay, with RIN ranging between 8.5 and 10.0.
|
| Label |
biotin / streptavidin-Cy3
|
| Label protocol |
For each sample 200 ng of total RNA were reverse transcribed, amplified by in vitro transcription and labelled with biotin-UTP using the Illumina Total Prep RNA amplification kit (IL1791, Ambion) following the manufacturer's instructions. Labelled sample quality was assessed by spectrophotometry and bioanalyzer. After preheating at 65ºC for 5 min.
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| |
|
| Hybridization protocol |
For each sample, 750 ng of biotinylated cRNA were hybridized in a BeadChip Hyb Chamber with rocking 16 h at 58ºC. The day after, the beadarrays were washed with Illumina proprietary washing solutions in a Hybex waterbath: first with static incubation for 10 min at 55ºC in E1BC solution, followed by 10 rinses by dipping in the same solution and shaking 5 min at 90 rpm in an orbital shaker; the next wash was by dipping 10 times in 100% ethanol and shaking 10 min at 110 rpm in an orbital shaker; this was followed by another wash in E1BC solution with 10 dippings followed by 2 min shaking at 90 rpm. Washed bead arrays were blocked in E1 buffer 10 min in rocking incubator and 10 additional minutes with 2 ml of E1 buffer plus streptavidin-Cy3. The fluorescent reagent was washed away with E1BC solution with 10 dippings plus 5 min shaking at 140 rpm. Finally beadarrays were dried by centrifugation 4 min at 275 rcf, followed by scanning in an Illumina Beadstation.
|
| Scan protocol |
The Beadscan software was used to control de scanner, generate .tif images and extract the raw data as tabulated text files. Default settings for DirectHyb Gene Expression were used.
|
| Description |
KO biological replicate 4; individual hippocampus sample from a knock out animal lacking PrPc protein expression because of a homozygous deletion of the Prnp gene
|
| Data processing |
The raw data was summarized per probe using BeadStudio software Gene Expression module and the summary data file was processed using the PILLA web interface tool (Lozano et al, unpublished), an implementation of the Lumi package (Du et al, 2008) developed within the Bioconductor project in the R statistical programming environment (Gentleman et al, 2004). Data were normalized using the rsn method and vst as the variance stabilization method. The log2 intensities were median centered and log ratios were computed as differences in log2 intensities for each probe. The SAM (significance analysis of microarrays) two class unpaired comparison test was applied with 300 permutations to detect statistically significant differences in gene expression between each of the three possible comparisons (Tusher et al, 2001).
|
| |
|
| Submission date |
May 26, 2009 |
| Last update date |
Jan 08, 2010 |
| Contact name |
Lauro Sumoy |
| E-mail(s) |
lsumoy@igtp.cat
|
| Organization name |
IGTP
|
| Department |
High Content Genomics and Bioinformatics
|
| Street address |
Ctra. Can Ruti, Camí de les escoles s/n
|
| City |
Badalona |
| State/province |
Barcelona |
| ZIP/Postal code |
08916 |
| Country |
Spain |
| |
|
| Platform ID |
GPL6105 |
| Series (1) |
| GSE16223 |
Prionic protein gene dosage is critical for maintenance of neuronal homeostasis of hippocampal circuits |
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