|
| Status |
Public on Jul 26, 2016 |
| Title |
X2 |
| Sample type |
RNA |
| |
|
| Source name |
Not stimulated DRG neurons
|
| Organism |
Mus musculus |
| Characteristics |
strain: NIH Swiss
age: E13.5
cell type: DRG neurons
stimulation pattern: not stimulated
|
| Treatment protocol |
DRG cultures were stimulated through platinum electrodes in contact with media on opposite sides of the barrier. Stimulation parameters and responses to stimulation have been reported previously (Eshete and Fields, 2001 and Fields et al, 1992). For this study, either 18 actions potentials every min (18/1) or 90 action potentials every 5 min (90/5) was delivered by a 6 V, 0.2 ms biphasic pulse for either 2hr or 5hr.
|
| Growth protocol |
Neurons were dissociated from spinal cords of 13.5-day old mouse embryos and plated at a density of 0.5 x 106 cells per ml into each side compartment in Eagle’s MEM containing 5% horse serum and 100 ng/ml nerve growth factor. Non-neuronal cell proliferation was inhibited by treatment with 13 μg/ml fluoro-2-deoxyurindine (Sigma, St. Louis, MO) by one day following plating for 5 days. Cultures were subsequently used for experiments 3- 4 weeks after which axons extend from the side compartments into the central compartment and could be electrically stimulated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was performed using RNAEasy Minikit (Qiagen, Germantown, MD, USA), following manufacturer's instructions. RNA concentration was determined with a Thermo Scientific NanoDrop ND-2000 and its quality with a 2100 Bioanalyzer (Agilent, DE)
|
| Label |
Cy5
|
| Label protocol |
Low Input Quick Amp labeling Kit two-color (Agilent, Cat # 5190-2306) that contains T7 RNA polymerase was used to simultaneously amplify total RNA and the positive controls (RNA Spike in Kit for Two Color, Agilent Cat # 5188-5279), and incorporate cyanine 3 (Cy3) or cyanine 5 (Cy5)-labeled CTP.
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| |
|
| Hybridization protocol |
825 ng from each of the Cy3 or Cy5 labeled samples from two biological replicas were mixed with Gene Expression Hybridization Kit (Agilent Cat # 5188-5242) and the final volume adjusted to 100 µL. The solution was slowly dispensed onto the gasket (Agilent Cat# G2534-60011) placed in the (Agilent) hybridization chamber, the (Agilent) mouse (4x44k 60 mer features, 4 microarrays per slide) chip with the active face downward was arranged over the gasket and the “sandwich” firmly closed. The samples were hybridized to the array at 65ºC for 17 h in an (Agilent) oven while vertically rotating (10 rot/min) to wet the gasket and asses the mobility of the bubbles.
|
| Scan protocol |
The hybridized chip was washed at room temperature using the Gene Expression Wash Pack (Agilent, Cat# 5188-5327) and Stabilization and drying solution (Agilent, Cat# 5185-5979) then immediately scanned with an Agilent G2565CA microarray scanner system with SureScan high resolution technology at 5µm pixel size and 20-bit scan mode.
|
| Description |
Gene expression in Not stimulated DRG neurons
Raw data file:
X1X2_US83000181_252665510921_S01_GE2_107_Sep09_1_2.txt
|
| Data processing |
The resulted Tagged Image File Format (tiffs) obtained through use of (Agilent) Scan Control Software 8.3 were analyzed with (Agilent) Feature Extraction vs.11 software. Data were normalized and analyzed with in-house developed algorithms described in Iacobas et al., Mol Genet Genomics 2012.
Data table contains normalized log2 ratio of the background subtracted signal of that feature and the median of the background subtracted signals of all valid features in the green or red channel of the array. A NULL indicates that the feature was not considered because is a control featrure, OR is locally corrupted, OR has saturated pixels, OR the median of the foreground signal is less than 2x median of the background signal
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| |
|
| Submission date |
Jul 26, 2016 |
| Last update date |
Jul 26, 2016 |
| Contact name |
Dumitru Andrei Iacobas |
| E-mail(s) |
daiacobas@pvamu.edu
|
| Phone |
936-261-9926
|
| Organization name |
Prairie View A&M University
|
| Department |
Undergraduate Medical Academy
|
| Lab |
Personalized Genomics
|
| Street address |
Ann Preston St
|
| City |
Prairie View |
| State/province |
TX |
| ZIP/Postal code |
77446 |
| Country |
USA |
| |
|
| Platform ID |
GPL10333 |
| Series (1) |
| GSE84872 |
Gene-regulatory networks activated by pattern-specific generation of action potentials in dorsal root ganglia neurons |
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