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Sample GSM187864 Query DataSets for GSM187864
Status Public on Aug 30, 2008
Title Mandibular E12.5 biological replicate 4
Sample type RNA
 
Source name Mandibular prominence tissue from mouse embryos at E12.5 days
Organism Mus musculus
Characteristics Strain: C57BL/6J
Age: embryonic day 12.5
Tissue: Mandibular prominence
Treatment protocol Embryos were dissected from the uterus in ice-cold DEPC-PBS medium and then individually placed into drops of DEPC-PBS media in a petri dish. Embryos were then staged by plug time and craniofacial features. After staging, each embryo was bisected with forceps at the level of the heart and the caudal portion was discarded. Tungsten needles were employed to isolate the combined maxillary and mandibular prominences and these were then separated into their individual components. Next, the medial and lateral frontonasal prominences were removed from the remainder of the head. Briefly, an incision was made in the ectoderm overlying the prominences to produce a flap of ectoderm that was peeled back with the loosely aggregated cells of the mesenchyme still attached. Note that if the cut was made too deep and extends into the forebrain region, loosely packed mesenchymal cells were no longer visible. These latter samples that were potentially contaminated with forebrain tissue were discarded. Suitably pure maxillary, mandibular, and frontonasal samples were placed in separate tubes containing RNAlater (Ambion) and stored at -20C for later pooling and processing. Pooling was necessary to obtain sufficient RNA for screening of the microarrays.
Growth protocol Following mating and visualization of a vaginal plug, pregnant females were sacrificed between E10 and E12.5 depending on the time-point under study. Mice employed for the E10, E11, and E12 timepoints were housed in a reverse light cycle room to facilitate dissections at midday for all samples.
Extracted molecule total RNA
Extraction protocol Tissue pools were dissolved in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's instructions and resuspended in a final volume of 100ul DEPC treated water. Subsequently, the sample was further purified using the RNeasy Mini Kit protocol (Qiagen, Inc.). The concentration of each total RNA sample was determined based on the absorbance at 260 nm (A260). The quality of each sample was first determined based on the ratio of A260 to A280 and samples in the range of 1.9-2.1 were considered adequately pure to be employed in cDNA synthesis. The integrity and purity of the individual samples from each prominence were also assessed using RT-PCR. Total RNA (1-2ug) was used for cDNA synthesis using random primers and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Subsequently, 2ul of the cDNA mixture was used for PCR in a 25ul reaction with Accuprime Taq DNA polymerase (Invitrogen) using the following conditions: 1 cycle at 94C for 3 min; 35 cycles at 94C for 45 sec, 58C for 45 sec, 68C for 1 min; and 1 cycle at 68C for 10 min. Specific primers were directed against transcripts that were diagnostic of the facial prominences including Tcfap2a, Dlx-2, Bmp2, and Gsc, or would detect contamination from adjacent tissue, e.g. Zic3 for forebrain tissue. Significantly contaminated or degraded RNA samples were discarded.
Label biotin
Label protocol Total RNA (2-5 ug) was converted to double-stranded cDNA (ds-cDNA) using the Superscript Choice System (Life Technologies, Inc.) and oligo-dT primer containing a T7 RNA polymerase promoter (Genset Corp.). After the second strand synthesis the reaction mixture was extracted with phenol chloroform-isoamyl alcohol and the ds-cDNA was recovered by ethanol precipitation. In vitro transcription was performed to generate biotin-labeled cRNA using an RNA Transcript Labeling Kit (Enzo. Inc.) and 3.3 uL ds-cDNA template in the presence of a mixture of biotin-labeled ribonucleotides. Biotin-labeled cRNA was then purified using an RNeasy affinity column (Qiagen). Next, the cRNA was fragmented to ensure optimal hybridization to the oligonucleotide array. Fragmentation was performed such that the cRNA fragments are between 35-200 bp in length by incubating the cRNA at 94C for 35 min in a fragmentation buffer. The sample was then added to a hybridization solution containing 100 mM MES, 1 M NaCl, and 20 mM EDTA in the presence of 0.01% Tween 20. The final concentration of the fragmented cRNA was 0.05 ug/uL. Labeled cRNA probes were generated from total RNA samples using the GeneChip Expression 3' Amplification One-Cycle Target Labeling Kit (Affymetrix, Santa Clara, CA).
 
Hybridization protocol Biotinylated cRNA probes in 200 ul sample buffer were hybridized to Affymetrix GeneChip Mouse 430 2.0 microarrays which contain probes corresponding to 39,000 mouse transcripts at 45C for 16 hr in a GeneChip Hybridization Oven 640 (Affymetrix, Santa Clara, CA.).
Scan protocol GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA.).
Description Mandibular prominence tissue from mouse embryos at E12.5 days, pooled from 3-4 embryos
Data processing The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
 
Submission date May 08, 2007
Last update date Aug 28, 2018
Contact name Trevor Williams
E-mail(s) trevor.williams@uchsc.edu
Phone 303-724-4571
Organization name UCHSC at Fitzsimons
Department Craniofacial Biology
Lab Trevor Williams
Street address MS-8120, PO Box 6511, 12801 E 17th Ave
City Aurora
State/province CO
ZIP/Postal code 80045
Country USA
 
Platform ID GPL1261
Series (1)
GSE7759 Spatial and Temporal Analysis of Gene Expression During Growth and Fusion of the Mouse Facial Prominences
Relations
Reanalyzed by GSE119085

Data table header descriptions
ID_REF
VALUE Signal
ABS_CALL indicating whether the transcript was present (P), absent (A), or marginal (M)
DETECTION P-VALUE

Data table
ID_REF VALUE ABS_CALL DETECTION P-VALUE
AFFX-BioB-5_at 175.209 P 0.00933744
AFFX-BioB-M_at 166.521 P 0.0166701
AFFX-BioB-3_at 93.8018 A 0.123572
AFFX-BioC-5_at 329.725 P 0.000389797
AFFX-BioC-3_at 205.955 P 0.000445901
AFFX-BioDn-5_at 597.792 P 0.00010954
AFFX-BioDn-3_at 2033.93 P 0.000856509
AFFX-CreX-5_at 9012.11 P 4.42873e-05
AFFX-CreX-3_at 11734.6 P 4.42873e-05
AFFX-DapX-5_at 10.8682 A 0.41138
AFFX-DapX-M_at 42.4962 A 0.195266
AFFX-DapX-3_at 4.34599 A 0.987453
AFFX-LysX-5_at 4.52923 A 0.617401
AFFX-LysX-M_at 11.0423 A 0.631562
AFFX-LysX-3_at 4.77735 A 0.48511
AFFX-PheX-5_at 2.30144 A 0.904333
AFFX-PheX-M_at 3.95652 A 0.760937
AFFX-PheX-3_at 55.3756 A 0.52976
AFFX-ThrX-5_at 5.46798 A 0.834139
AFFX-ThrX-M_at 25.241 A 0.382599

Total number of rows: 45101

Table truncated, full table size 1375 Kbytes.




Supplementary file Size Download File type/resource
GSM187864.CEL.gz 4.2 Mb (ftp)(http) CEL

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