Transcriptional profiling of light response in the zebrafish at several organizational levels: the whole animal, the organ and the cell. Exposure of larvae, heart organ cultures and cell culture cells to light pulses of 1 and 3 hours duration and measurement of changes in gene expression compared to controls kept in the dark.

Zebrafish Light Pulse

The light induced transcriptome of the zebrafish

Affymetrix GeneChip Zebrafish Genome Array [Zebrafish]

The Light Responsive Transcriptome of the Zebrafish: Function and Regulation.

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Transcriptional profiling of light response in the zebrafish at several organizational levels: the whole animal, the organ and the cell. Exposure of larvae, heart organ cultures and cell culture cells to light pulses of 1 and 3 hours duration and measurement of changes in gene expression compared to controls kept in the dark.


P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MTAB-17036

Adult zebrafish (Tübingen strain) were raised according to standard methods. Fertilized eggs were collected within 2 h of laying, and aliquots of 20 eggs were transferred into 20 ml of E3 buffer in 25-cm2 tissue culture flasks. The flasks were incubated in a large-volume thermostat-controlled water bath for 5 days in constant darkness at 25 C, then illuminated with a tungsten light source for 1 or 3 hours or maintained in the dark before RNA extraction.

P-MTAB-17037

Freshly dissected tissue was placed in L15 medium supplemented with 15% fetal calf serum and with gentamycin, streptomycin and penicillin. Organs were dissected from fish and washed 4 times with medium (10 ml/5 hearts). 5 hearts each were placed into cell culture flasks containing 5 ml of medium and submerged in the water bath in constant darkness for 4 days, then subjected to the light pulse as done for the larvae and directly processed for RNA extraction.

P-MTAB-17038

PAC2 cell culture was carried out essentially as described. Confluent cells were split 1:6 into 75cm2 flasks and submerged in the waterbath in constant conditions for a week before light pulse treatment and RNA isolation.

P-MTAB-17039

Exposure to light pulses of 1 and 3 hours duration and measurement of changes in gene expression compared to controls kept in the dark.

P-MTAB-17040

Total RNA was extracted from triplicate samples using Trizol RNA isolation reagent (GIBCO-BRL, San Diego, California, United States) according to the manufacturer's instructions.

P-MTAB-17041

Synthesis and labeling of antisense RNA was performed as recommended by the array manufacturer, using kits for double-stranded cDNA synthesis (Invitrogen, Carlsbad, CA, USA), for transcription and labeling of antisense RNA (Enzo Life Sciences, Farmingdale, NY, USA) and for probe purification and hybridization controls (Affymetrix, Santa Clara, CA, USA).

P-MTAB-17042

Background correction, normalization and probe set summarization were performed using the robust multi array algorithm with background adjustment (gcrma, Wu, 2004).

TREATMENT

Myoblasts were separated by magnetic cell sorting according p75NTR expression. Both negative and positive fractions were collected. p75NTR expression was monitored by Real-Time PCR immediately after selection and reached 10fold difference in the two preparations. Selection was repeated four times on the same cell line and reached similar purity.

HYBRIDIZATION

Standard Illumina hybridization protocol.

SCANNING

Scanning in the Beadstation 500 (Illumina Inc) was performed according to standard Illumina protocols.

Zebrafish Light Pulse

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