|
| Status |
Public on Aug 01, 2017 |
| Title |
P45 D-1 |
| Sample type |
RNA |
| |
|
| Source name |
Medial entorhinal cortex, deep layers, P45, replicate 1
|
| Organism |
Rattus norvegicus |
| Characteristics |
age: P45
layer: Deep
strain: Long Evans
|
| Treatment protocol |
Long Evans rats were deeply anesthetized and sacrificed by guillotine. The brain was immediately placed in oxygenated ACSF, sliced on a microtome (400μm) and placed in RNAlater. Layer slices were then dissected under a microscope.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Ambion's RNAqueous®-Micro kit, following the manufacturer's recommendations except for an extra 1 minute incubation with wash buffer, and the ethanol added for precipitation was increased to 1.25x lysis buffer volume to include small RNAs. RNA was eluted with 2x10μl RNAqueous Elution Buffer. The RNA in four samples (RS-198_20, RS-198_25, RS-198_27, RS-198_28) was extracted using Ambion's mirVana kit according to the manufacturer's recommendations and eluted in 2x50 µl mirVana Elution Buffer. The amount of RNA in all samples was analyzed on the Nano Drop, and the quality of the RNA on the BioAnalyzer Nano Chip
|
| Label |
Cy3
|
| Label protocol |
100 ng of total RNA from each sample was spiked with MicroRNA Spike-In Kit (Agilent Technologies), treated with calf intestinal phosphatase, and labeled with Cy3 using the miRNA Complete Labeling and Hyb Kit according to the manufacturer's recommendations.
|
| |
|
| Hybridization protocol |
The Cy3-labeled miRNA samples were hybridized for 20hrs at 55°C onto separate Rat miRNA Microarrays, Release 15.0 (Agilent Technologies, AMADID 029200, 8x15K format). The microarrays were then washed with increasing stringency using Gene Expression Wash Buffers (Agilent Technologies) followed by drying with acetonitrile (SIGMA)
|
| Scan protocol |
Fluorescent signal intensities were detected with Scan Control A.8.4.1 Software (Agilent Technologies) on the Agilent DNA Microarray Scanner
|
| Description |
miRNA expression in entorhinal cortex deep layers at postnatal age P45
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) to obtain QC stats of Feature Outliers, Background and Non-Uniformity. The additive error was below 12 for all samples, and the Hyb Spike-In signal was above the recommended threshold of 2.50 for all arrays.
The raw values were processed using the AgiMicroRna Bioconductor package, filtering according to the quality flag (filterMicroRna function with settings wellaboveNEG = FALSE, limIsGeneDetected = 50, limNEG = 25), and normalizing using the quantile method.
|
| |
|
| Submission date |
Aug 17, 2016 |
| Last update date |
Aug 01, 2017 |
| Contact name |
Lene Christin Olsen |
| E-mail(s) |
[email protected]
|
| Organization name |
Norwegian University of Science and Technology - NTNU
|
| Department |
Medical Faculty
|
| Street address |
Erling Skjalgsons gt. 1
|
| City |
Trondheim |
| ZIP/Postal code |
7491 |
| Country |
Norway |
| |
|
| Platform ID |
GPL19562 |
| Series (1) |
| GSE85753 |
miRNA expression in rat medial entorhinal cortex layers during postnatal development |
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