|
| Status |
Public on Nov 29, 2021 |
| Title |
Neurons, DMSO, replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
Primary cultures of cortical neurons were generated from forebrains of embryonic (E) 17.5 mice and incubated with DMSO (0.1%) as solvent control for 8 h.
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Primary cultures of cortical neurons
treatment: DMSO control
|
| Treatment protocol |
Staurosporine (Merck #S6942, Kenilworth, NJ, USA) was added to Neurobasal Medium (Gibco #21103-049, Thermo Fisher Scientific, Waltham, MA, USA) to reach a final concentration of 1 M. Whole medium from primary neuron cell cultures was removed and replaced by staurosporine-containing medium. Primary neuron cultures were incubated with staurosporine for 8 h. Control samples were incubated with DMSO (0.1%) as solvent for 8 h.
|
| Growth protocol |
Primary cultures of cortical neurons were generated from forebrains of embryonic (E) 17.5 mice, and seeded into a 24-well plate at a density of 5x105 cells/well and incubated for 3 d at 37˚C in humidified air with 5% (v/v) CO2, before starting the experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
miRNA extraction from conditioned medium and cortical neurons was performed as previously described using organic extraction followed by mirVana™ isolation kit (Thermo Fisher Scientific #AM1560, Waltham, MA, USA) application. 1 ml of cell-conditioned medium was used for organic total RNA extraction. 2x106 cells grown on a 6-well plate were used for organic extraction from neurons. Cells were lysed prior to organic extraction.
|
| Label |
biotin
|
| Label protocol |
For miRNA expression analysis small RNA was biotin labeled using the FlashTag™ HSR Biotin RNA labeling kit for Affymetrix miRNA arrays (Genisphere LLC., Hatfield, PA, USA), according to manufacturer's protocol and detected by fluorescent emission. The labeling kit using a two-step process that does not require any purification prior to array hybridization. Small RNA was subjected a tailing reaction followed by ligation of the biotinylated template to the target RNA.
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| |
|
| Hybridization protocol |
The labeled RNA sample was then added to a hybridization mixture and hybridized to Affymetrix GeneChip miRNA microarrays at 48°C for 16 hrs, washed and stained accordingly to the manufacturer’s protocol (Hybridization Wash and Stain Kit, Affymetrix Inc., Santa Clara, CA, USA)
|
| Scan protocol |
Arrays were scanned with a GeneChip scanner 3000 G7 (Affymetrix).
|
| Description |
small RNA
Gene expression data from neurons incubated with DMSO, replicate 1
|
| Data processing |
Microarray data were processed using the robust multi-array average (RMA) method on the R/Bioconductor platform using default parameter (i.e. quantile normalisation and RMA background correction). For annotation, the Bioconductor package pd.mirna.4.0 was used.
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| |
|
| Submission date |
Jan 21, 2021 |
| Last update date |
Nov 29, 2021 |
| Contact name |
Matthias Futschik |
| E-mail(s) |
mfutschik@ualg.pt
|
| Organization name |
Universidade do Algarve
|
| Lab |
SysBioLab
|
| Street address |
Campus de Gambelas
|
| City |
Faro |
| ZIP/Postal code |
8005-139 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL19117 |
| Series (1) |
| GSE165263 |
miR profiling of apoptotic murine neurons and their supernatent |
|
