Affymetrix GeneChip Zebrafish Genome Array [Zebrafish]

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Zebrafish embryo animal cap explants were differentiated by injection of vhnf1 mRNA into the one-cell stage embryo, and treatment of excised explants with applied FGF8 protein.  To identify unique targets of the combination of vhnf1+FGF8, each factor was applied alone and in combination, and differences in gene expression used to identify unique targets of vhnf1+FGF8.

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Transcription profiling of animal caps from Danio rerio embryos expressing GFP or vhnf1 and treated with FGF8 or BSA protein

Zebrafish embryo animal cap explants were differentiated by injection of vhnf1 mRNA into the one-cell stage embryo, and treatment of excised explants with applied FGF8 protein.  To identify unique targets of the combination of vhnf1+FGF8, each factor was applied alone and in combination, and differences in gene expression used to identify unique targets of vhnf1+FGF8.


Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-29990

Following the recommended protocol from Affymetrix: "GeneChip Eurkaryotic Small Sample Target Labeling Assay Version II".  Briefly: First round amplification about 100ng of total RNA was reverse transcribed using T7-Oligo(dT) promoter primer and SuperScriptII reverse transcriptase.  Second strand cDNA was synthesized with DNA Polymerase I, in the presence of RNaseH and DNA ligase.  double stranded cDNA was purified by ethanol precipitation.  cDNA was transcribed using random primers and RNA polymerase, and the resulting RNA was purified using RNeasy Mini Kit and protocol.  First round amplification should yield about 1-2 ug of cRNA.   Second round amplification and labeling of cRNA about 300 ug cRNA from first round amplification was used in a second reverse transcription reaction, using random primers and SuperScript II reverse transcriptase.  again, second strand cDNA was synthesized with DNA Polymerase I, and resulting cDNA was purifed by ethanol precipitation.  Finally, in vitro transcription using the ENZO BioArray HighYield RNA Transcript Lebeling Kit was performed, to produce biotin labeled cRNA probe.  This product was cleaned up over RNeasy columns (QIAGEN).

P-MEXP-9893

Animal cap explants were excised essentially according to Grinblat et al. (1999.  Methods in Cell Biology, volume 59, pp.127-156), with minor modifications.  In brief, embryos at very late blastula (40% epiboly)/early gastrula (germ ring) stages (5 hours post-fertilization), were dechorionated and then grouped into sets of four embryos in a dish coated with 1% agarose and filled with 1XMBS (Modified Barth's Saline).  A glass injectin needle was used to cut a square piece of tissue off the top (animal cap) of the embryo, while the embryo was held with forceps.  Explants from all four embryos were pushed together and allowed to heal into a single ball of cells.  These 4X explant tissue pieces were transferred to 4-well Nuclon delta Surface tissue culture plates, up to 5 tissue fragments per well, in 1XMBS.  

P-MEXP-9896

Zebrafish embryos were injected at the one-cell stage with mRNA encoding vhnf1 or gfp (control).  Explants containing these mRNAs were isolated at the early gastrula stage (see Isolation protocol), and grown in 1XMBS at 29 deg. C (see Growth protocol).  At the equivalent of shield stage, protein was added to media in which explants were grown:  FGF8b at 1 ug/mL plus BSA at 1 mg/mL, or BSA alone (control).  Explant aggregates were treated with vhnf1+FGF8, vhnf1+BSA, gfp+FGF8 or gfp+BSA, and then were incubated for about 5 hr longer at 29 deg. C, until the 3-somite stage equivalent.  

P-MEXP-9897

Samples were collected into a single tube (see Pooling protocol), and 100 uL TRIZOL reagent (Invitrogen) was added.  Samples were frozen to -80 degrees C for at least 12 hours, and then thawed.  20 ug glycogen added, and RNA was isolated following manufacturer's instructions.  Precipitated RNA was resuspended in 50 uL water, and re-purified using the QIAGEN RNeasy Micro kit, following manufacturer protocols.  

Transcription profiling of animal caps from Danio rerio embryos expressing GFP or vhnf1 and treated with FGF8 or BSA protein

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