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Sample GSM1289544 Query DataSets for GSM1289544
Status Public on Jul 31, 2016
Title 59 (T2_Hg)
Sample type RNA
 
Source name Hg treatment, T2
Organism Anguilla anguilla
Characteristics tissue: liver
agent: Hg treatment
time: T2 (6 days)
Treatment protocol An exposure laboratory experiment was performed in the “Ur Biologia eta Experimentazio Zerbitzua” (UBEZ) of the University of the Basque Country (UPV/EHU) A total of 84 eels, 20.23 ± 2.84 cm in length, were acclimatized to laboratory conditions for 2 weeks in a 10 L water glass tanks under controlled conditions: 12 h light/dark cycle at 18oC, conductivity 600 μS. During this period animals were fed with commercial flakes. Ammonium, nitrites and nitrates kits (Sera GmbH) were used to systematically asses the levels of nitrogenous compounds which were maintained at 0-0.5mg/l; 0-0.5 mg/l, and 5-10 mg/l, respectively. When highest concentrations within those ranges were achieved seawater was partially replaced. A day before exposure experiment, animals were placed in 4 aquaria with 8 L of water at 600 μS. In each aquarium 21 eels were randomly distributed and exposed to: metal-mercury (Hg) (Fluka-Sigma-Aldrich) at a concentration of 100μg/L; and to a phitosterol, β-sitosterol (Sigma) at a concentration of 150 μg/ L. The last compound needed ethanol (0.01%) as vehicle. In addition to an ethanol control group, a water control (600μS) without contaminant was also prepared.
Extracted molecule total RNA
Extraction protocol Liver (100 mg) was gridded in TRizol® (Invotrgen) using Precellys homogenizer (Bertin technologies, France) and Precellys lysing kit MK28-R (Bertin; 2*5000, 30sec). After that, RNA was extracted following TRIzol® (Invitrogen) manufacturers´s instructions and purified using RNAsy Kit (Qiagen) with the additional DNAse treatment (Qiagen) on columns. RNA quantity and quality was measured using Epoch TM Take 3 TM spectrophotometer (Biotek) and 2100 bioanalyzer (Agilent) respectively. RIN values above 8 were discharged.
Label Cy3
Label protocol The hybridization step was performed using the One-Color Microarray-Based Gene Expression Analysis “Low Input Quick Amp Labeling” protocol (Agilent, version 6.5, May, 2010) following manufacturer’s instructions. Total RNA was used as starting material to produce cRNA, which was in vitro synthesized using QuickAmp Labelling Kit one-color (Agilent). At first step, it was retrotranscribed using AffinityScript reverse transcriptase, an MMLV reverse transcriptase and Oligo dT primers coupled to T7 promoter. After that, in vitro transcription and Cy-3 labelling of ds cDNA was performed using the antisense T7 Promoter Primer, T7 RNA Polymerase and Cy-3-CTP.
 
Hybridization protocol All cRNA labelled samples purified and quantified using RNeasy spin columns (Qiagen) and NanoDrop ND-1000 spectophotometer respectively. As a result yield and specific activity of each reaction was measured and all of them were above following limits 0.825 ug yield and 6 pmol Cy3 per μg of cRNA. Finally, 50 ng of labelled cRNA were obtained. Hybridization step was performed in a SureHyb hybridization chamber (Agilent), a total of 600ng of labelled cRNA was used in a volume of 40 μL. They were incubated in the hybridization oven at 10 rpm, 65 oC for 17 hours. Slides were washed using GE wash buffer 1 (1 minute), GE wash buffer 2 (37 oC, 1 minute), and acetonitrile wash (10 seconds). Finally, scanning process was performed on a G2565 DNA microarray scanner (Agilent) with ozone barrier slide cover using following settings: Green channel dye, 61*21.6 mm scan region, scan resolution of 3 μm and 20 bit Tiff.
Scan protocol Feature Extraction software v. 10.7.3.1 (Agilent) was used to feature signal intensity extraction. It was subsequently processed on GeneSpring GX 12.6 (Agilent Technologies).
Data processing As preprocessing, first all Feature Extraction flags were transformed to Absent/Marginal/Present calls (Compromized/Not Detected/Detected Flags in the new GeneSpring versions), as follows: Feature is NOT Positive and significant: ABSENT (Compromized);Feature is NOT uniform: ABSENT (Compromized);Feature is NOT well above background: MARGINAL (Not Detected);Feature is Saturated: MARGINAL (Not Detected); Feature is Population outlier: ABSENT (Compromized);Threshold raw signals (lineal data) to 1. After that a geometric mean of replicated probes was calculated and the resulted Raw Signal was transformed as Log2. Furthermore, an intra-chip normalization was done based on quantile normalization. Finally a baseline was performed with the mean value of control samples.
 
Submission date Dec 16, 2013
Last update date Jul 31, 2016
Contact name Ibon Cancio
E-mail(s) [email protected]
Organization name UPV/EHU
Street address B/sarriena s/n
City Leioa
ZIP/Postal code 48940
Country Spain
 
Platform ID GPL18071
Series (1)
GSE53344 Hepatic transcription profile study on European eels (Anguilla anguilla) exposed to Hg and b-sitosterol

Data table header descriptions
ID_REF
VALUE RMA normalized signal intesities

Data table
ID_REF VALUE
CUST_4145_PI426546238 -0.9445205
CUST_16103_PI426544517 -0.5515771
CUST_7271_PI426544517 0.24929428
CUST_3322_PI426546238 0.001653671
CUST_8034_PI426546238 0.13491201
CUST_8652_PI426544517 0.9599619
CUST_10181_PI426546238 -0.09328461
CUST_11936_PI426546248 -0.059664726
CUST_18116_PI426546248 0.36930752
CUST_2689_PI426546248 0.6306195
CUST_1124_PI426544517 -1.3380651
CUST_2294_PI426547332 -0.013201714
CUST_11934_PI426546248 -3.70471
CUST_14249_PI426544517 0.8438597
CUST_11899_PI426544517 -0.57684803
CUST_1975_PI426546238 -0.018233776
CUST_8559_PI426546238 0.07695007
CUST_8457_PI426544517 0.059583664
CUST_15866_PI426546248 0.18675804
CUST_20810_PI426544517 1.1632948

Total number of rows: 45543

Table truncated, full table size 1484 Kbytes.




Supplementary file Size Download File type/resource
GSM1289544_253514710011_201307170351_S01_GE1_107_Sep09_1_4.txt.gz 9.0 Mb (ftp)(http) TXT
Processed data included within Sample table

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