Affymetrix GeneChip Mouse Expression Array MOE430A [MOE430A]

Affymetrix GeneChip Chicken Genome Array [Chicken]

Affymetrix GeneChip Mouse Genome 430 2.0 [Mouse430_2]

Affymetrix GeneChip Zebrafish Genome Array [Zebrafish]

Evolutionary plasticity of segmentation clock networks.

Evolutionary plasticity of segmentation clock networks

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4 microarray time series was generated to identify cyclic genes of the segmentation clock in the mouse (2 time series), the chicken and the zebrafish. The right posterior half presomitic mesoderms (PSM) from 20 mouse embryos, 18 chicken embryos and 21 zebrafish embryos were dissected while the contralateral side of the embryo containing the left PSM was immediately fixed to be analyzed by in situ hybridization using a Lfng (fot mosue and chicken) or hes7 (zebrafish) probe to order the samples along the segmentation clock oscillation cycle. Probes were produced from RNA extracted from each of the dissected posterior half PSMs using a two-step amplification protocol and were hybridized to Affymetrix GeneChip MOE430A, MOE430 2.0, Affymetrix GeneChip chicken genome array,  or Affymetrix GeneChip zebrafish array.

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Comparative transcription profiling of mouse, chicken and zebrafish in the presomitic mesoderms to identify cyclic genes of the segmentation clock

Divergent cyclic genes but conserved oscillatory signaling pathways in vertebrate segmentation clocks

4 microarray time series was generated to identify cyclic genes of the segmentation clock in the mouse (2 time series), the chicken and the zebrafish. The right posterior half presomitic mesoderms (PSM) from 20 mouse embryos, 18 chicken embryos and 21 zebrafish embryos were dissected while the contralateral side of the embryo containing the left PSM was immediately fixed to be analyzed by in situ hybridization using a Lfng (fot mosue and chicken) or hes7 (zebrafish) probe to order the samples along the segmentation clock oscillation cycle. Probes were produced from RNA extracted from each of the dissected posterior half PSMs using a two-step amplification protocol and were hybridized to Affymetrix GeneChip MOE430A, MOE430 2.0, Affymetrix GeneChip chicken genome array,  or Affymetrix GeneChip zebrafish array.


P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MTAB-17414

2 days old chicken embryos (15 or 16 somite stages, 12HH) were dissected in cold Tyrode solution made with DEPC treated water in RNAse free conditions. After being cut out of the egg, the embryos were pinned in a silicone-covered petridish, ventral side up and treated with pancreatin (4xUSP from Invitrogen). The endoderm was removed, and the right posterior half of the PSM (determined with the help of a reticule) was separated from the surrounding structures with a tungsten needle

P-MTAB-17415

right PSM (posterior half) were dissected in cold Tyrode's solution prepared in RNAse-free conditions.  The posterior half PSM was dissected using tungsten needles to remove the surrounding structures (e.g., the ectoderm, neural tube, endoderm and lateral plate) and stored in Trizol (Invitrogen) at -80C  for RNA extraction.

P-MTAB-17416

Total RNA was isolated from each dissected posterior half PSM  using Trizol.

P-MTAB-17417

RNA was extracted from the dissected PSM piece and amplified before hybridization of each sample individually on an Affymetrix GeneChip array. RNA samples extracted from mouse were amplified either using the first cycle of the GeneChip Eukaryotic small sample target labeling Assay version II, and the second cycle of the Two-Cycle Eukaryotic Target Labeling Assay, described in the Affymetrix, GeneChip® Expression Analysis, Technical Manual rev5 for samples hybridized to A-AFFY-23. mouse, chicken and zebrafish samples hybridized to A-AFFY-45, A-AFFY-103, or A-AFFY-39 where amplified and labelled usingthe Two-Cycle Eukaryotic Target Labeling Assay, described in the Affymetrix, GeneChip® Expression Analysis, Technical Manual rev5 . Total RNA was extracted from chicken and zebrafish PSM pieces using Trizol.

P-MTAB-17418

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

P-TABM-808

CD-1 mouse embryos (9.0 days post-coitus [dpc]) were dissected in cold Tyrode's solution prepared in RNAse-free conditions. Mouse embryos from each litter were dissected one by one, while the rest of the embryos were kept on ice. No more than three embryos per litter were dissected in order to limit the time between removal of the uterus and embryo dissection to reduce the risk of RNA degradation. The time spent for dissection per embryo was limited to 15-20 min. After tail bud removal, the right posterior side was cut out and treated with dispase (2 mg/mL) for 1 min prior to dissecting the posterior half presomitic mesoderm (PSM). The posterior half PSM was dissected using tungsten needles to remove the surrounding structures (e.g., the ectoderm, neural tube, endoderm and lateral plate) and stored in Trizol (Invitrogen) at -80C  for RNA extraction.

P-TABM-810

Amplification of the samples was performed as described in GeneChip Eukaryotic small sample target labeling Assay version II. Total RNA from each sample was used to generate first-strand cDNA by using a T7-linked oligo (dT) primer. After second-strand synthesis, in vitro transcription was performed with unlabelled ribonucleotides yielding 1.2 to 2.4 ug of cRNA. Using 400ng of cRNA from the first round of amplification, a second round of cDNA synthesis was then performed followed by in vitro transcription with biotinylated UTP and CTP (Enzo BioArray high-yield RNA transcript labeling kit) yielding 65 to 93 ug of biotinylated cRNA. RNA quality was confirmed after each round of amplification by A260/A280 ratio (> 2.0) and Bioanalyzer analysis (mean size transcripts 2Kb after 1st round and 1Kb after 2nd round). The biotin-labeled cRNA samples were fragmented to sizes < 200bp (checked by Bioanalyzer).

Comparative transcription profiling of mouse, chicken and zebrafish in the presomitic mesoderms to identify cyclic genes of the segmentation clock

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