|
| Status |
Public on Dec 01, 2014 |
| Title |
PCB_50 - Aa29 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Fish from laboratory PCBs experiment
|
| Organism |
Anguilla anguilla |
| Characteristics |
source location: laboratory
agent: PCB
dose: [50ng/g PS]_diet
tissue: Caudal fin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was carried out by means of the SV total RNA islotaion Kit, promega). Sampleswere placed in 450 µl of RNA lysis (Promega) buffer in sterile microtube and were homogenized by means of two metal beads and tissue homogenizer (Retsch). After 2 min of centrifugation at 10,000 rpm, 400 µl of the lysate was recovered. One volume of Phenol:Chloroform:Isoamyl Alcohol 25:24:1 was then added. The homogenate was vortexed 10s and centrifuged 10 min at RT. The upper phase was recovered. One volume of 75% EtOH was adeed. After homogenization by vortexing, the mix was transferred to a spin column and the standard manufacturer’s procedures were followed (including DNAseI treatment).. Quality and integrity of the total RNA were controlled by electrophoresis on a 1% agarose gel.
|
| Label |
Cy3
|
| Label protocol |
For the first step of reverse-transcription procedure, 10µg of RNA was mixed with 3µg of random primer and 2µg of oligodT. Water was added for 20µl qs. Mix was incubated 5 min at 70°C followed by10 min at 18°C and finally stored at 4°C. 20µl of the RT mix (GoScript™ 5X Reaction Buffer: 8µl; DTT [0.1M]: 4µl; MgCl2 [25mM]: 4.8µl; 50X aa-dNTP mix (25mM each): 1µl; dUTP: 1µl; GoScript™ RT: 2µl) was added to the RNA mix. The mix was incubated during 12h at 42°C followed by 5 min at 18 °C and stored at 4°C. RNAs were then hydrolyzed with 4µl of EDTA [0.2M] pH 8 and 6µl of NaOH [1M] during 10 min at 70°C. To re-adjust the pH, 6µl of HCL [1M] was then added. cDNA purification was carried out with the Qiagen PCR purification kit following the manufacturer’s protocol. cDNA was eluted in 60µl of sodium bicarbonate [0.1M] pH 9 and placed at -20°C before labeling. In order to normalize microarray data, we used a common reference design. This was composed by a pool of 30 wild eels of Certes (15 fishes collected in 2011 and 15 in 2012). This reference was combined in equal amounts with each sample before to be hybridized on the microarray slide. . Reference and sample cDNAs were labeled with CyDye™ Post-Labelling Reactive Dye Pack (Amersham). Each cDNA sample and reference was mix with 1 vial of CyDye (Cy3 for sample and Cy5 for reference) and incubated at RT in the dark during 90 min. Reaction was stopped by adding 15µl of hydroxylamine hydrochloride [4M] and incubated during 15 min in the dark. Purification of labeled cDNA was carried out with the Qiagen PCR purification kit following the manufacturer’s protocol and eluted in 60µl of water. Amount of labeled cDNA as well as the frequency of incorporation (FOI) were determined spectrophotometrically (Epoch, Biotek).
|
| |
|
| Channel 2 |
| Source name |
reference RNA
|
| Organism |
Anguilla anguilla |
| Characteristics |
reference composition: a pool of 30 wild eels of Certes (15 fishes collected in 2011 and 15 in 2012)
tissue: liver
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was carried out by means of the SV total RNA islotaion Kit, promega). Sampleswere placed in 450 µl of RNA lysis (Promega) buffer in sterile microtube and were homogenized by means of two metal beads and tissue homogenizer (Retsch). After 2 min of centrifugation at 10,000 rpm, 400 µl of the lysate was recovered. One volume of Phenol:Chloroform:Isoamyl Alcohol 25:24:1 was then added. The homogenate was vortexed 10s and centrifuged 10 min at RT. The upper phase was recovered. One volume of 75% EtOH was adeed. After homogenization by vortexing, the mix was transferred to a spin column and the standard manufacturer’s procedures were followed (including DNAseI treatment).. Quality and integrity of the total RNA were controlled by electrophoresis on a 1% agarose gel.
|
| Label |
Cy5
|
| Label protocol |
For the first step of reverse-transcription procedure, 10µg of RNA was mixed with 3µg of random primer and 2µg of oligodT. Water was added for 20µl qs. Mix was incubated 5 min at 70°C followed by10 min at 18°C and finally stored at 4°C. 20µl of the RT mix (GoScript™ 5X Reaction Buffer: 8µl; DTT [0.1M]: 4µl; MgCl2 [25mM]: 4.8µl; 50X aa-dNTP mix (25mM each): 1µl; dUTP: 1µl; GoScript™ RT: 2µl) was added to the RNA mix. The mix was incubated during 12h at 42°C followed by 5 min at 18 °C and stored at 4°C. RNAs were then hydrolyzed with 4µl of EDTA [0.2M] pH 8 and 6µl of NaOH [1M] during 10 min at 70°C. To re-adjust the pH, 6µl of HCL [1M] was then added. cDNA purification was carried out with the Qiagen PCR purification kit following the manufacturer’s protocol. cDNA was eluted in 60µl of sodium bicarbonate [0.1M] pH 9 and placed at -20°C before labeling. In order to normalize microarray data, we used a common reference design. This was composed by a pool of 30 wild eels of Certes (15 fishes collected in 2011 and 15 in 2012). This reference was combined in equal amounts with each sample before to be hybridized on the microarray slide. . Reference and sample cDNAs were labeled with CyDye™ Post-Labelling Reactive Dye Pack (Amersham). Each cDNA sample and reference was mix with 1 vial of CyDye (Cy3 for sample and Cy5 for reference) and incubated at RT in the dark during 90 min. Reaction was stopped by adding 15µl of hydroxylamine hydrochloride [4M] and incubated during 15 min in the dark. Purification of labeled cDNA was carried out with the Qiagen PCR purification kit following the manufacturer’s protocol and eluted in 60µl of water. Amount of labeled cDNA as well as the frequency of incorporation (FOI) were determined spectrophotometrically (Epoch, Biotek).
|
| |
|
| |
| Hybridization protocol |
For cDNA hybridization, microarray was pre-hybridized in 5X SSC buffer; 0.1% SDS; 0.1 g.L-1 BSA during 45 min at 55°C. Microarray was washed twice 5 min in 0.1X SSC at RT followed by 30s in purified water under continuous agitation. Microarray was then centrifuged 2 min for drying. 800ng of cDNA sample was pooled with 800ng of cDNA reference and hybridized on microarray in 2X hybridization buffer (8X SSC; 0.4% SDS; 0.1g.L-1 calf thymus DNA) and incubated at 55°C in Agilent chamber overnight. Hybridized microarray was washed at 55°C in 2X SSC; 0.1% SDS buffer, washed, twice 5 min in 2X SSC; 0.1% SDS buffer under agitation at RT; twice 5 min in 0.1X SSC; 0.1% SDS buffer and finally 4 times in 0.1X SSC buffer and dried by centrifugation (2 min).
|
| Scan protocol |
Microarray data were generated by the Innoscan 710 microarray scanner using Mapix software.
|
| Description |
PCB [50ng/g PS]_diet
|
| Data processing |
The filtering of raw data was done for both green and red channels. The median foreground (F635median and F532median) and median background (B635median and B532median) was chosen for the data normalization. If the ratio Fmedian/Bmedian was lower than 1.5, data were removed from the analysis. For each channel, the background was removed of the foreground data and the mean of triplicates (three spots for each gene) was then calculated and both green and red channels were used for data normalization. Normalization and statistical analysis was done by using the BRB-arrayTools version 4.4.0 software package (http://linus.nci.nih.gov/BRB-ArrayTools.html). Each array was median normalized over entire arrays and genes were excluded if 50% of values were missing in the complete set of arrays.
Data were normalized by median-centering log(red/green)-ratios in each array, based on the average of the red and green log-intensities.
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| |
|
| Submission date |
Nov 04, 2014 |
| Last update date |
Dec 01, 2014 |
| Contact name |
lucie baillon |
| Organization name |
université de Bordeaux UMR ePOC
|
| Street address |
place du docteur Peyneau
|
| City |
Arcachon |
| ZIP/Postal code |
33120 |
| Country |
France |
| |
|
| Platform ID |
GPL19017 |
| Series (1) |
| GSE62954 |
Detecting the effects of Cd and PCBs by means of a non-invasive transcriptomic approach in laboratory- and wild-contaminated European eels (Anguilla anguilla) |
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