|
| Status |
Public on Nov 30, 2020 |
| Title |
Ipsilateral cervical DRGs, Stretch-induced axon growth, rep 5 |
| Sample type |
RNA |
| |
|
| Source name |
Rat DRG, 0 % strain 5 days, 25 % strain 8 days in vitro
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague dawley
developmental stage: Embryonic day 15.5 + 13 days in vitro
tissue: Cervical dorsal root ganglia
|
| Treatment protocol |
Following seeding, and growth of axons across the substrates, mechanotransduced cultures underwent 25 % daily cumulative strain until a maximum stretch-growth rate of 3 mm/day was reached and sustained for 2 days.
|
| Growth protocol |
C1-C7 cervical DRG explants were bilaterally isolated from E15.5 rat embryos removed from a single timed-pregnant Sprague-Dawley rat and seeded into matching stretch and sham cultures. DRGs were seeded onto 1.5 x 2.5 cm Aclar (Honeywell, Morristown NJ) manipulating substrates which overlaid a large Aclar stationary substrate. Explants were seeded individually utilizing a stereo microscope along the edges of the manipulating substrates; ensuring outgrowth of all explants onto the underlying substrates. Aclar substrates consisted of 2.00 mil (51 μm thickness) UltRx 2000 Aclar washed with 50 % acetone-soaked cotton swabs and rinsed thoroughly 2 days prior to coating. On the day of seeding, substrates were coated with high molecular weight poly-d-lysine (354210, Becton Dickinson, Bedford MA) at 60 μg/cm2 in PBS w/Ca2+ & Mg2+ for 1 hour at 37°C. Culture media consisted of Neurobasal (Gibco 21103) supplemented with 2 % B-27 (Gibco 17504), 1 % FBS-HI, 20 ng/mL NGF (Gibco 13290-010), 0.5 mM L-glutamine (Gibco 25030081), 2.5 g/L D-glucose (Sigma G-7528), 20 µM FdU (Sigma F-0503), and 20 µM Uridine (Sigma U-3003), which was changed the day after seeding and every 3 to 4 days thereafter.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Messenger RNA was isolated by detaching the Aclar substrates containing DRG explants and immersing directly into microcentrifuge tubes containing lysis buffer. Utilizing a standard silica-based RNA isolation kit (RNeasy, Qiagen, Valencia CA), DNA was digested on-column and eluted mRNA was frozen.
|
| Label |
Biotin
|
| Label protocol |
cDNA was generated from total RNA using the Ambion WT Expression Kit according to the manufacturer's protocol. The cDNA was fragmented and end labeled following the GeneChip Whole Transcript Terminal Labeling and Hybridization protocol.
|
| |
|
| Hybridization protocol |
The Affymetrix GeneChip WT Terminal Labeling and Hybridization protocol was used following the format for Array type 169 to hybridize cDNA to the arrays for 16 hours at 45C in the GeneChip Hybridization Oven 640.
|
| Scan protocol |
Affymetrix GCS 3000 Scanner w/AGCC v1.1.0.845 software
|
| Description |
Gene expression data from cultured DRG neurons undergoing stretch-mediated axon growth
|
| Data processing |
Partek Genomic Suite software (Partek Inc., St. Louis, MO) was used to analyze the array data. The arrays were normalized using the robust multi-array average (RMA) algorithm and principal component analysis was performed to check for chip outliers. Fold changes were calculated and a paired two-tailed t-test was performed to generate p-values. Up and downregulation of genes in stretch-grown samples relative to shams were expressed as positive and negative changes, respectively. In addition to p-values, Significance Analysis of Microarrays (SAM) v4.0 was used to generate q-values (Tusher et al., 2001). Normalized data from Partek was analyzed pairwise using SAM to generate q-values for all genes.
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| |
|
| Submission date |
May 04, 2017 |
| Last update date |
Nov 30, 2020 |
| Contact name |
Joseph R. Loverde |
| E-mail(s) |
axonstretch@me.com
|
| Organization name |
New Jersey Institute of Technology
|
| Department |
Department of Biomedical Engineering
|
| Lab |
Center for Injury Biomechanics, Materials, and Medicine
|
| Street address |
111 Lock Street
|
| City |
Newark |
| State/province |
NJ |
| ZIP/Postal code |
07103 |
| Country |
USA |
| |
|
| Platform ID |
GPL6247 |
| Series (1) |
| GSE98535 |
Transcriptional Changes in Rat Dorsal Root Ganglia Neurons During Stretch-Mediated Axon Growth |
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