Affymetrix GeneChip Zebrafish Genome Array [Zebrafish]

Aryl Hydrocarbon Receptor  Activation Produces Heart-Specific Transcriptional and Toxic Responses in Developing Zebrafish

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For analysis of gene expression changes in the zebrafish larvae heart in response to TCDD exposure, three replicate samples of heart tissue were collected at 73, 74, 76 and 84 hours post fertilization from larvae exposed to 1 ng/ml TCDD or vehicle from 72 - 73 hours post fertilization. For analysis of gene expression changes in the extracardiac tissue in response to TCDD exposure, three replicate samples of zebrafish larvae bodies with the heart tissue removed were collected at 73, 74, 76 and 84 hours post fertilization from larvae exposed to 1 ng/ml TCDD or vehicle from 72 - 73 hours post fertilization.

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Transcription profiling time series of zebrafish hearts treated with dioxin (TCDD)

For analysis of gene expression changes in the zebrafish larvae heart in response to TCDD exposure, three replicate samples of heart tissue were collected at 73, 74, 76 and 84 hours post fertilization from larvae exposed to 1 ng/ml TCDD or vehicle from 72 - 73 hours post fertilization. For analysis of gene expression changes in the extracardiac tissue in response to TCDD exposure, three replicate samples of zebrafish larvae bodies with the heart tissue removed were collected at 73, 74, 76 and 84 hours post fertilization from larvae exposed to 1 ng/ml TCDD or vehicle from 72 - 73 hours post fertilization.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-26870

Fertilized cmcl2::GFP +/- embryos were obtained from adult cmcl2::GFP+/+ males (AB background) bred with AB females, maintained at 27 degrees Celcius with a 14-h/10-h light/dark cycle in egg water (60 microgram/ml Instant Ocean Salts, Aquarium Systems)in plastic petri dishes. Each day non-viable embryos were removed from dishes and half of the water replaced with fresh egg water.

P-MEXP-26871

Exposure: Hatches larvae were statically exposed to 2,3,7,8-tetrachlorodibenzo-p-dixon (TCDD, >99% purity from Chemsyn, Lenexa, KS) for 1 hr from 72 to 73 hours post fertilization by maintaining larvae in egg water containing either vehicle (0.1% dimethyl sulfoxide) or 1 ng/ml TCDD. Exposures were carried out in tightly capped glass vials (130 larvae in 10 ml dosing solution) on a gently shaking platform.   Heart Extraction & Collection: Hearts were simultaneously extracted from 130 zebrafish larvae from the same dosing vial via shear forces applied by passing the larvae through a needle and syringe. Larvae were gently anesthetized with Tricaine-S, placed in a microfuge tube with Lebovitz's L15 media + 10% fetal bovine serum (FBS) and mounted on a ring stand. A 5-cc syringe with a 19 gauge needle was mounted so the tip of the needle was near the bottom of the microfuge tube. The syringe plunger was pulled up and pushed down at a constant rate of one push or pull per second for 1 minute. The sheared larvae were removed from the microfuge tube and dripped over a 105 micron mesh filter with retained the bodies allowed the hearts to pass through. The filtrate containing the hearts was then dripped over a 40 micron mesh filter that retained the hearts and permitted smaller debris tissue to pass through. The hearts, expressing green fluorescent protein, were rinsed from the 40 micron mesh filter into a 60-mm petri dish with fresh media, then located with epifluoresence and immediately collected into a clean microfuge tube on ice. Samples were centrifuged at 3000 x g for 5 minutes to pellet the hearts. The supernatant was removed, and the pelleted hearts were snap frozen in liquid nitrogen and stored at -80 degrees Celcius. During each heart extraction session about 80-90 hearts were recovered from the 130 larvae exposed to TCDD or vehicle in the same dosing vial.

P-MEXP-26873

For each treatment replicate, 6 heart extraction sessions were carried out to generate 6 blocks of 80-90 hearts. The 6 blocks of 80-90 hearts for each treatment replicate were pooled during the initial steps of the RNA extraction to generate one extractant for each treatment replicate.  

P-MEXP-26875

Total RNA was isolated from ~500 hearts for each treatment replicate using a QIAGEN RNeasy Mini Kit (QIAGEN, Valencia, CA). Each treatment replicate consisted of 6 blocks of 80-90 hearts, pelleted and frozen. The heart tissue in each block was homogenized in lysis buffer (supplied with kit) and applied to a QIAshredder column (QIAGEN, Valencia, CA). The QIAshredder elutants from each of the 6 blocks were applied to one RNA extraction column to pool the sample blocks. The columns were washed and the total RNA eluted in nuclease-free water according the manufacturer's instructions.

P-MEXP-26880

Total RNA was isolated from 20 zebrafish larvae bodies without heart tissue for each treatment group using a QIAGEN RNeasy Mini Kit (QIAGEN, Valencia, CA). The body tissue was homogenized in lysis buffer (supplied with kit) and applied to a QIAshredder column (QIAGEN, Valencia, CA). The QIAshredder elutant was applied to an RNA extraction column, and the columns were washed and the total RNA eluted in nuclease-free water according the manufacturer's instructions.

P-MEXP-26951

Exposure: Hatches larvae were statically exposed to 2,3,7,8-tetrachlorodibenzo-p-dixon (TCDD, >99% purity from Chemsyn, Lenexa, KS) for 1 hr from 72 to 73 hours post fertilization by maintaining larvae in egg water containing either vehicle (0.1% dimethyl sulfoxide) or 1 ng/ml TCDD. Exposures were carried out in tightly capped glass vials (130 larvae in 10 ml dosing solution) on a gently shaking platform.   Extracardiac Body Tissue Collection: Hearts were simultaneously extracted from 130 zebrafish larvae from the same dosing vial via shear forces applied by passing the larvae through a needle and syringe. Larvae were gently anesthetized with Tricaine-S, placed in a microfuge tube with Lebovitz's L15 media + 10% fetal bovine serum (FBS) and mounted on a ring stand. A 5-cc syringe with a 19 gauge needle was mounted so the tip of the needle was near the bottom of the microfuge tube. The syringe plunger was pulled up and pushed down at a constant rate of one push or pull per second for 1 minute. The sheared larvae were removed from the microfuge tube and dripped over a 105 micron mesh filter with retained the bodies allowed the hearts to pass through. Twenty bodies with hearts removed were collected into a microfuge tube on ice. Excess fluid was removed and the tissue snap frozen in liquid nitrogen and stored at -80 degrees Celcius.   

Transcription profiling time series of zebrafish hearts treated with dioxin (TCDD)

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