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| Status |
Public on Aug 17, 2011 |
| Title |
Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes. |
| Organism |
Rattus norvegicus |
| Experiment type |
Expression profiling by array
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| Summary |
Identifying the early gene program induced by GnRH would help understand how GnRH-activated signaling pathways modulate gonadotrope secretory response. We previously analyzed GnRH-induced early genes in LbT2 cells, however these lack GnRH self-potentiation, a physiological attribute of gonadotropes. To minimize cellular heterogeneity, rat primary pituitary cultures were enriched for gonadotropes by 40-60% using a sedimentation gradient. Given the limited number of gonadotropes, RNA was amplified prior to microarray analysis. Thirty-three genes were up-regulated 40 minutes after GnRH stimulation. Real-time PCR confirmed regulation of several transcripts including fosB, c-fos, egr-2 and rap1b, a small GTPase and member of the Ras family. GnRH stimulated rap1b gene expression in gonadotropes, measured by a sensitive single cell assay. Immunocytochemistry revealed increased Rap1 protein in GnRH-stimulated gonadotropes. These data establish rap1b as a novel gene rapidly induced by GnRH and a candidate to modulate gonadotropin secretion in rat gonadotropes.
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| Overall design |
Primary rat gonadotrope cells were exposed to 10 nM GnRH for 40 min, then harvested and processed for RNA extraction using a Qiagen RNeasy mini kit (Qiagen, Valencia, CA). A total of 12 Affymetrix Rat Expression Array 230 v2.0, namely 6 GnRH-treated and 6 vehicle-treated samples, each containing 31,000 gene clusters, were used. Data analysis was performed by Affymetrix GeneChip Operating System (GCOS). A gene was considered to be up-regulated by GnRH if there is at least 50% concordance across multiple pairwise comparisons of GnRH- vs. vehicle-treated microarrays, and if the fold-change was at least 1.50.
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| Contributor(s) |
Yuen T, Pincas H |
| Citation(s) |
22127306 |
| Submission date |
Aug 16, 2011 |
| Last update date |
Jul 31, 2017 |
| Contact name |
Tony Yuen |
| E-mail(s) |
tony.yuen@mssm.edu
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| Organization name |
Icahn School of Medicine at Mount Sinai
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| Street address |
One Gustave L. Levy Place
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platforms (1) |
| GPL1355 |
[Rat230_2] Affymetrix Rat Genome 230 2.0 Array |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA145721 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE31399_RAW.tar |
16.7 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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