Total RNA was extracted using an RNA Trizol B kit (Teltest, Paisley, Scotland). Forty-five embryos or larvae were homogenized with 4.5 mL RNA Trizol B reagent. The homogenate was mixed with 200 μL chloroform, shaken gently for 15 s, and kept on ice for 5 min before centrifugation at 11,000 rpm for 10 min at 4 °C. An aliquot of 600 μL of supernatant was mixed well with 600 μL isopropanol and 3 M acetic acid, and stored at 15 °C for 10 min before further centrifugation at 11,000 rpm for 10 min at 4 °C. The supernatant was removed, and the precipitate was washed with 75% ethanol. The precipitate was allowed to dry for 30 min and was dissolved again in an adequate amount of diethylpyrocarbonate (DEPC, Sigma) to obtain total RNA. The RNA extract was dried at 37 °C for 5~10 min before measuring its total RNA content in a spectrophotometer (Hitachi U-2000, Japan) at OD 260/280 nm.
Label
Alexa Flour 647
Label protocol
cDNA probes were synthesized by reverse transcription of 10 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen) and were labeled with Alexa Flour 647 (cold-treatment groups) and Alexa Flour 555 (control groups) (Invitrogen).
Total RNA was extracted using an RNA Trizol B kit (Teltest, Paisley, Scotland). Forty-five embryos or larvae were homogenized with 4.5 mL RNA Trizol B reagent. The homogenate was mixed with 200 μL chloroform, shaken gently for 15 s, and kept on ice for 5 min before centrifugation at 11,000 rpm for 10 min at 4 °C. An aliquot of 600 μL of supernatant was mixed well with 600 μL isopropanol and 3 M acetic acid, and stored at 15 °C for 10 min before further centrifugation at 11,000 rpm for 10 min at 4 °C. The supernatant was removed, and the precipitate was washed with 75% ethanol. The precipitate was allowed to dry for 30 min and was dissolved again in an adequate amount of diethylpyrocarbonate (DEPC, Sigma) to obtain total RNA. The RNA extract was dried at 37 °C for 5~10 min before measuring its total RNA content in a spectrophotometer (Hitachi U-2000, Japan) at OD 260/280 nm.
Label
Alexa Flour 555
Label protocol
cDNA probes were synthesized by reverse transcription of 10 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen) and were labeled with Alexa Flour 647 (cold-treatment groups) and Alexa Flour 555 (control groups) (Invitrogen).
Hybridization protocol
The zebrafish 14K OciChip array chip was pretreated with 1% bovine serum albumin (BSA) (fraction V), 4x saline-sodium citrate (SSC), and 1% sodium dodecylsulfate (SDS) at 42 °C for 45 min, and then hybridized overnight in a cocktail containing 5x Denhardt's solution, 6x SSC, 0.5% SDS, 50% formamide, 50 mM sodium phosphate, and 2 µg/µl yeast tRNA. Slides were washed with 2x SSC and 0.1% SDS (for 5 min), 1x SSC and 0.1% SDS (for 5 min), 0.5x SSC (for 5 min), and twice with 0.1x SSC (for 2 min each).
Scan protocol
Scanning was performed with a Genepix scanner (Molecular Devices, Sunnyvale, CA). The acquired images were analyzed using Genepix and Genespring software (Aglient Technologies, Foster City, CA). The measurements of spots were filtered by flags, and Lowess normalization was performed after subtracting the median background.
Description
The commercial zebrafish 14K oligonucleotide set (MWG Biotech AG, Ebersbach, Germany) was obtained and printed on UltraGAPS-coated slides (Corning, New York, NY) with an OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI) according to the manufacturer’s instructions. The 14,067 oligonucleotides represented 9666 genes (7009 singlet genes and 2657 redundant genes), and the redundancy of the chip was 31%. A detailed description of the oligonucleotide information can be obtained on the Ocimun Biosolution website (http://www.ocimumbio.com/web/default.asp).
Data processing
The experiment contained 3 biological replicates with different samples. Differentially expressed genes were selected from those with at least 2 of 3 significant signals (with a ratio > 2 or < 0.5), and then the Significant Analysis of Microarray method (SAM 3.02) was used to determine statistical significance.
