|
| Status |
Public on Nov 11, 2014 |
| Title |
Wildtype sample at 17.5 d.p.c, biological rep1 (MOE430A) |
| Sample type |
RNA |
| |
|
| Source name |
Mouse hindquarters at 17.5 d.p.c
|
| Organism |
Mus musculus |
| Characteristics |
tissue: embryo hindquarters
genotype: Wildtype C57BL/6J mice
age: 17.5 d.p.c
|
| Treatment protocol |
Three individual animals of a single genotype were pooled to give five pools (N=15, total number of arrays=5) for each genotype/timepoint combination for three tissues: whole embryos (13.5 d.p.c.), hindquarters (17.5 d.p.c.), or pectoralis muscle (d35). Mice were reared, sacrificed by CO2 asphyxiation (embryonic timepoints) or cervical dislocation (d35) and tissues were flash-frozen in liquid nitrogen as previously described in Steelman et al., 2006. FASEB J. Mar;20(3):580-2.
|
| Growth protocol |
Tissues were collected from mouse whole embryos (13.5 d.p.c.), hindquarters (17.5 d.p.c.), or pectoralis muscle (d35). After collection, muscles were flash-frozen in liquid nitrogen as previously described in Steelman et al., 2006. FASEB J. Mar;20(3):580-2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The frozen tissues from whole embryo or hindquarter only were immersed in RLT lysis buffer (Qiagen, Valencia, CA) and homogenized for 45 seconds, then centrifuged at 3600g for 10 min to pellet cell debris. Total RNA was isolated using the RNase Midi Kit (Qiagen) with on-column DNase digestion according to manufacturer's protocol. Isolation from pectoralis muscle was conducted as described in Steelman et al., 2006. FASEB J. Mar;20(3):580-2.
|
| Label |
biotin
|
| Label protocol |
Double-stranded cDNA was synthesized from pooled RNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). T7-oligo(dT) primer and SuperScript II reverse transcriptase were used in first-strand synthesis. Biotin-labeled cRNA was generated with the BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY). cDNA and cRNA cleanup, as well as fragmentation of cRNA, were carried out using the GeneChip Sample Cleanup Module (Qiagen, distr. by Affymetrix).
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| |
|
| Hybridization protocol |
Target RNA was hybridized to both chip A and chip B of the GeneChip Mouse Expression Set 430 (MOE430; Affymetrix, Santa Clara, CA). The A and B chips correspond to GPL339 and GPL340, respectively. Hybridization was carried out according to the GeneChip Expression Analysis Technical Manual (Affymetrix).
|
| Scan protocol |
Scanning was carried out according to the GeneChip Expression Analysis Technical Manual (Affymetrix).
|
| Description |
Gene expression data from hindquarters at 17.5 d.p.c
|
| Data processing |
Robust multi-array analysis (RMA) (Irizarry et al., 2003. Biostatistics 4: 249-264) was performed to normalize the raw data by using R/affy package (Gautier et al., 2004. Bioinformatics 20: 307-315).
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| |
|
| Submission date |
Nov 10, 2014 |
| Last update date |
Nov 11, 2014 |
| Contact name |
James M Reecy |
| Organization name |
Iowa State University
|
| Department |
Animal Science
|
| Lab |
Reecy Lab
|
| Street address |
2255 Kildee Hall, Iowa State University
|
| City |
Ames |
| State/province |
IA |
| ZIP/Postal code |
50011-3150 |
| Country |
USA |
| |
|
| Platform ID |
GPL339 |
| Series (1) |
| GSE63154 |
Gene co-expression network analysis of myostatin regulation at three different mouse developmental timepoints |
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