Strain: C57BL/6J Age: embryonic day 11.0 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 E11.0 days, pooled from 8-9 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.