|
| Status |
Public on Feb 15, 2021 |
| Title |
AD11_hp_1m_sample2 |
| Sample type |
RNA |
| |
|
| Source name |
hippocampus
|
| Organism |
Mus musculus |
| Characteristics |
tissue: brain
transgenic: anti-NGF antibody, heavy+light chain
Sex: female
age: 1 month
subject_id: ad17
|
| Treatment protocol |
The moce did not undergo any treatment before tissue extraction.
|
| Growth protocol |
AD11 transgenic mice (Ruberti et al., 2000) express a recombinant version of the monoclonal antibody mAb alfaD11 that specifically recognizes and neutralizes NGF (Cattaneo et al., 1988; Covaceuszach et al., 2008). The AD11-VH controls are transgenic mice where only the heavy chain of the transgenic antibody is expressed, while in AD11 mice express the whole functional antibody (heavy + light chain). AD11-VH mice were used as transgenic control mice, since they are consistently negative with respect to all the neurodegeneration markers. Each AD11 or AD11-VH mouse was individually tested by transgene genotyping. Mice were kept under a 12 hours dark to light cycle, with food and water ad libitum. Experiments were performed according to the European law for laboratory animal welfare and experimentation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hippocampus of the right hemisphere was dissected from the brains of freshly sacrificed mice. Total RNA was isolated from these brain areas using Trizol (Invitrogen) and DNAse treated by Qiagen columns. Quality and integrity of each sample was checked using the Agilent BioAnalyzer 2100 (Agilent RNA 6000 nano kit): only samples that showed a ratio of absorbance 1.8< OD260/OD280 <2.0 and OD260/OD230 >2.0 were selected samples with a RNA Integrity Number (RIN) index higher than 8.0 .
|
| Label |
Cy3
|
| Label protocol |
All the experimental steps involving the labelling, hybridization and washings of the samples were done following the Agilent protocol (http://chem.agilent.com). The Small RNA kit was used for separation and quantification of miRNA. One-color miRNA expression was performed according to the Agilent procedure, version 1.5, December 2007 (http://www.chem.agilent.com). Labeled miRNAs were obtained from 100 ng of total RNA through the ligation of a 5′-cytidine bisphosphate-Cy3 (pCp-Cy3, Agilent Technologies) group at the 3′-end of each miRNA. To enhance the T4 RNA-ligase (Ambion) efficiency, we had previously treated total RNA with alkaline phosphatase (TaKaRa) at 37°C for 30 min. Labeled miRNAs were purified on chromatography columns (Micro Biospin 6, Biorad Laboratories) and then hybridized on a microarray. Each slide contained 8 identical microarrays with probes for 567 mouse and 73 mouse viral miRNAs.
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| |
|
| Hybridization protocol |
Hybridizations were performed at 55°C for 20 h in a rotating oven. The hybridized microarrays were disassembled at room temperature in Agilent Gene Expression Wash Buffer 1. After the disassembly, the microarrays were washed in Gene expression Buffer 1 for five minutes at room temperature, followed by washing with Gene Expression Wash Buffer 2 for five minutes at 37°C. The microarrays were then treated with Acetonitrile for five minutes at room temperature.
|
| Scan protocol |
Post-hybridization image acquisition was accomplished using the Agilent Scanner G2564B. Data extraction from the 20 bits TIFF images was accomplished by Agilent Feature Extraction (FE) ver 10.7 software using the standard Agilent one-color microRNA gene expression extraction protocol (ver miRNA_107_Sep09 ).
|
| Description |
none
|
| Data processing |
Data analyses pre-processing was performed using the proprietary Agilent GeneSpring GX software. Samples were normalized by the software to the 75th percentile in Log2 scale. The “gTotalGeneSignal” column of raw data files has been background subtracted and outlier rejected and is appropriate for use as the measured intensity for each miRNA gene without further manipulation. “gTotalGeneSignal” has then been summarized by gene symbol and the final normalized aggregated expression data in Log2 scale are provide in the *GeneView.txt files. Differential espressed miRNAs have been selected by R-Bioconductor using a combination of two thresholds: |Log2 fold change| >1.0 and 1-tail heteroscedastic t-test p-value <0.05.
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| |
|
| Submission date |
Dec 04, 2020 |
| Last update date |
Feb 15, 2021 |
| Contact name |
Ivan Arisi |
| E-mail(s) |
i.arisi@ebri.it
|
| Phone |
+39-06-49255230
|
| Organization name |
European Brain Research Institute
|
| Department |
Bioinformatics Facility
|
| Street address |
viale Regina Elena 295
|
| City |
Roma |
| ZIP/Postal code |
00161 |
| Country |
Italy |
| |
|
| Platform ID |
GPL8824 |
| Series (1) |
| GSE162675 |
Nerve Growth Factor neutralization promotes oligodendrogenesis by increasing miR-219a-5p levels |
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