|
| Status |
Public on Jul 01, 2011 |
| Title |
2B1_Dlx5_replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
2B1 Otic vesicle cell line over-expressing Dlx5 and GFP
|
| Organism |
Mus musculus |
| Characteristics |
tissue: 2B1 cell line
genotype/variation: N/A
age: 4 days post-differentiation in-vitro
|
| Treatment protocol |
Otic vesicles were directly transferred into 1ml of TRIZOL. The 2B1 cell line was allowed to proliferate at 32oC in the presence of gamma-interferon to a confluency of 70-80% and then transfected and shifted to 37oC with no gamma-interferon to induce differentiation for 4 days after which total RNA was isolated.
|
| Growth protocol |
Otic vesicles from wild-type and Dlx5-null embryos were microdissected from mouse developmental stages E10 and E10.5 in ice-cold PBS (calcium and magnesium free). These were timed-pregnancies determined by a vaginal plug, and each embryo was staged independently in a litter by the number of somites. Once the vesicles were isolated as cleanly as possible from the embryos, they were placed in 1mg/ml solution of dispase dissolved in PBS (calcium and magnesium free) for 10-15 minutes on ice to facilitate the removal of closely attached mesenchyme. For each stage and genotype two biological replicates were collected, each comprising between 4 and 8 otic vesicles. For the 2B1 cell line, two independent cultures were used for both the empty vector-transfected and the Dlx5- transfected samples. Cells were grown in chick embryo fibroblast medium at 32oC to a confluency of 70-80% in the presence of gamma-interferon, then transfected and shifted to 37oC with no gamma-interferon to induce differentiation for 4 days after which total RNA was isolated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Biotin
|
| Label protocol |
To synthesize biotin-labeled target (cRNA) for hybridization to Affymetrix arrays, 22ul of the purified cDNA were used as template in in-vitro transcription reactions using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Life Sciences, NY). Besides the template cDNA, all other components were added following the instructions provided by the manufacturer, and reactions were incubated at 37oC for 10 hours on the PCR machine mentioned above under 'extract protocol'. Labeled cRNA was purified using an RNA purification kit (Qiagen) following the manufacturer’s instructions and eluted in 50ul of water.
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| |
|
| Hybridization protocol |
Fragmentation of cRNA was carried out following the standard Affymetrix procedure, and 10ug of fragmented cRNA from each sample was hybridized to the Mouse Genome 430 2.0 gene chips for 16-18 hours at 45oC.
|
| Scan protocol |
GeneChips were scanned using GeneChip Scanner 3000.
|
| Description |
2B1 Otic vesicle cell line transiently transfected with Dlx5 and GFP expression vector and differentiated for 4 days at 37oC without gamma-interferon, replicate2
|
| Data processing |
Analysis was carried out in Expression Console software (Affymetrix) by employing the Robust Multichip Analysis (RMA) algorithm. All normalized (log-base-2) expression values below 5.5 were considered ‘Absent’. For cell line microarray data the cut-off for making ‘Present’ calls was set to 4.8 since this was the expression of Dlx5 in Dlx5-transfected cells.
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| |
|
| Submission date |
Jun 16, 2010 |
| Last update date |
Jul 01, 2011 |
| Contact name |
Samin Sajan |
| Organization name |
University of Chicago
|
| Street address |
920 E. 58th Street, CLSC 317
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE22381 |
Identification of downstream transcriptional targets of Dlx5 during early mouse inner ear (otocyst/otic vesicle) development |
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