cell line: ZF4 cells infection: IPNV time point: 6 h.p.i.
Biomaterial provider
Academia Sinica
Treatment protocol
The E1S virus was propagated in ZF4 cell monolayer at a 5 multiplicity of infection (MOI) per cell. Infected cultures were incubated at 18°C and collected sample at 6hr post-infection.
Extracted molecule
total RNA
Extraction protocol
ZF4 cells were seeded in a 100-mm petri dish and cultivated for more than 24 h. These cells were then infected with MOI of 5 of IPNV and incubated for 6h. At the completion of each incubation period, the culture medium was aspirated, and cells were wished with PBS. The total RNAs of ZF4 cells were also extracted with TRIzol reagent (Invitrogen) and were further purified by On-Column RNase-free DNase digestion (QIAGEN) to remove possible genomic DNA contamination. The RIN value of RNA samples before being applied to microarray was measured to be 10.0 by Agilent 2100 Bioanalyzer to confirm the RNA quality.
Label
Cy5
Label protocol
cDNA probes were synthesized by reverse transcription of 10 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen).The amine-modified DNA then was incubated with resuspend one aliquot of Cy5 Dye (Amersham Bioscience, Buckinghamshire, UK) at RT in the dark for overnight. Finally, the probe was purified by kit and was eluted with elution EB buffer.
ZF4 cells were seeded in a 100-mm petri dish and cultivated for more than 24 h. At the completion of each incubation period, the culture medium was aspirated, and cells were wished with PBS. The total RNAs of ZF4 cells were also extracted with TRIzol reagent (Invitrogen) and were further purified by On-Column RNase-free DNase digestion (QIAGEN) to remove possible genomic DNA contamination. The RIN value of RNA samples before being applied to microarray was measured to be 10.0 by Agilent 2100 Bioanalyzer to confirm the RNA quality.
Label
Cy3
Label protocol
cDNA probes were synthesized by reverse transcription of 10 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen).The amine-modified DNA then was incubated with resuspend one aliquot of Cy3 Dye (Amersham Bioscience, Buckinghamshire, UK) at RT in the dark for overnight. Finally, the probe was purified by kit and was eluted with elution EB buffer.
Hybridization protocol
Amino-allay dye coupling was carried out using the SuperScript Plus Indirect cDNA Labeling System (Invitrogen) according to the manufacture’s instructions. We have optimized a reverse transcription labeling protocol using 40 μg total RNA, 5 μg of anchored oligo(dT) primer ((dT)20VN) and SuperScript III reverse transcriptase. After 3 hour incubation at 46°C, the reaction was stopped by incubating at 70°C for 15 min in the presence of 1N NaOH, and then neutralized the solution by adding 1N HCl. Bring the reaction mixture to a final volume of 100 μl with nuclease-free water. Purify the amine-modified DNA using a MinElute PCR Purification Kit (QIAGEN), following the instructions in the kit, except perform twice washes instead of one and elute the probe by 0.1M NaHCO3. Resuspend one aliquot of Alexa Flour Dye in 20 μl aa-cDNA labeled probe and then incubate at RT in the dark for overnight. Repeat the purification by MinElute PCR Purification kit and elute with elution EB buffer. Yeast tRNA (10 μg) were added to the sample, dried in a speedvac at 45°C and redissolved in 70 μl of hybridization buffer (formamide-based) purchased from MWG (Germany) . The mixture was denatured at 95°C for 2 min. The solution was collected by brief centrifugation and applied onto the oligo area on the microarray slides. Coverslips were applied (60 x 22 mm) and then immersed the chamber in a water bath for hybridization overnight at 42°C. Arrays were washed by 2x SSC, 0.1% SDS, followed by 1x SSC, 0.1% SDS, followed by 0.5 x SSC, followed by 0.1 x SSC only at room temperature for 5 min each time. Slides were subsequently dried by brief centrifugation. Arrays were scanned using an Axon GenePix 4000B scanner and median spot intensities collected using Axon GenePix Pro5.1 (Molecular Devices, Sunnyvale, CA)
Scan protocol
Scanning was performed with a ScanArray Gx scaner (PerkinElmer, Waltham, MA). The acquired images were analyzed using ScanArray Express 3.0 (PerkinElmer)and Genespring software (Aglient Technologies, Foster City, CA).
Description
Zebrafish ZF4 cell line derived from 24-hpf zebrafish embryos was purchased from the American Type Culture Collection (CRL-2050) and cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and penicillin-streptomycin. The isolated virus, E1-S, a member of the Ab strain of IPNV, was isolated from Japanese eels in Taiwan. The commercial zebrafish 14K oligonucleotides set (MWG Biotech AG, Ebersbach, Germany) were obtained and were printed on UltraGAPS Coated slide (Corning, New York, NY ) with use of the OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI) according to the manufacture’s instructions. The 14,067 oligonucleotides represent 9666 genes (7009 singlet genes and 2657 redundant genes), and the redundancy of this chip is 31 %.
Data processing
Data files were imported into GeneSpring GX 7.3 (Agilent Technologies, Foster City, CA) for further analysis. ‘LOWESS Normalization’ was applied for data normalization for GeneSpring. Expression data sets must pass all the following quality control categories before they are used for cluster analysis. First, the hybridization results were not flagged as bad. Second, net intensities of both channels were equal to or greater then 500. Third, statistical analyses were applied to the triplicate data for each spot and repeat three times. For expression data, ratio values equal to or greater than 2 were considered up-regulated or down-regulated.
