|
| Status |
Public on May 31, 2019 |
| Title |
Liver_Control_21d rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Liver, Control 21d
|
| Organism |
Danio rerio |
| Characteristics |
tissue: Liver
gender: Male
|
| Treatment protocol |
Samples were waterborne exposed to 0.01 mg/L of maltose-coated Ag NPs of 20 nm or ionic silver for 3 and 21 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted following the TRizol® extraction method (ThermoFisher Scientific). Concentration of RNA was measured in a Biophotometer (Eppendorf, Hamburg, Germany). RNA was purified with RNeasy mini kit (Qiagen, Venlo, The Netherlands). In addition, RNA quality was assessed in an Agilent 2100 Bioanalyzer (Agilent Technologies). Only RNA samples with a RIN value above 8.1 were used for microarray and qPCR analysis.
|
| Label |
Cy3
|
| Label protocol |
Amount of nucleic acid labeled: 100 ng. Agilent Protocol One-Color Microarray-Based Gene Expression analysis (Low Input Quick Amp Labeling) Version 6.5 May 2010. Samples are retrotranscribed (first strand synthesis) and labeled using Low imput Quick Amp Labeling kit, One color (Agilent Technologies, Cat. Nº 5190-2305) following manufacturer instructions. First, total RNA is retrotranscribed with AffinityScript Reverse Transcriptase (AffinityScript RT), an engineered thermostable mutant derived from Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, using Oligo dT primers coupled to T7 promoter. Double stranded cDNA synthesized by AffinityScript RT is in vitro transcribed by T7 RNA pol in the presence of either Cy3-CTP or Cy5-CTP fluorophores to generate amplified and labeled cRNA. Labeled samples are purified with silica-based RNeasy spin columns (Qiagen). After labeling and purification, cRNA is quantify with NanoDrop ND-1000 spectrophotometer in order to determine the yield and specific activity of each reaction. Yield: µg of cRNA. Should be > 1.65 µg. Specific activity: pmol Cy3 per µg of cRNA. Should be >6. All the labeled samples where above those limits.
|
| |
|
| Hybridization protocol |
Chamber type: SureHyb hybridization chamber (Agilent). Quantity of each labeled extract used: 1.65 ƒÝg. Volume: 100 uL. Temperature (oC): 65. Duration: 17 hours at 10 rpm in the hybridization oven.
|
| Scan protocol |
Scanned on a G2565CA DNA microarray scanner (Agilent Technologies), with ozone-barrier slide covers (Agilent P/N G2505-60550) SCAN C SETTINGS Dye Channel Scan RegionGreen 61 x 21,6 mm Scan resolution (um) 6 Tiff 20 bit
|
| Data processing |
Images (.tiff images) were quantified using Agilent Feature Extraction Software (ver. 10.7.3.1) (Agilent Technologies). The Feature Extraction software (FE) extracts the raw data from Cy3 (Green(g) channel) fluorescence signal (mean signal) from the spot which contains the probes (control features and biological or non control features) and from background. After processing of the raw signal it applies several algorithms for the calculation of the processed signal, which will be used in the analysis, and other useful data for the assessment of the quality of the processed data. The processed signal is corrected for background and microarray processing artifacts (Spatial detrending). For background correction it uses the negative control feature data, which corresponds to sequences with no expected hybridization, and it is an indication of unspecific binding or hybridization. More detailed data processing description is available on the series record.
|
| |
|
| Submission date |
Nov 10, 2016 |
| Last update date |
May 31, 2019 |
| Contact name |
José María Lacave |
| E-mail(s) |
josemaria.lacave@ehu.es
|
| Organization name |
University of the Basque Country
|
| Department |
Zoology and Animal Cell Biology
|
| Street address |
Sarriena s/n
|
| City |
Leioa |
| State/province |
Bizkaia |
| ZIP/Postal code |
E-48940 |
| Country |
Spain |
| |
|
| Platform ID |
GPL14664 |
| Series (1) |
| GSE89756 |
Bioaccumulation, cellular and molecular effects in adult zebrafish after exposure to cadmium sulphide quantum dots and ionic cadmium |
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