Samples were dietary exposed to 0.001 mg/L and 0.1 mg/L of PVP-PEI-Ag NPs of 5 nm for 3 and 21 days
Growth protocol
Zebrafish Wild type AB Tübingen
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. Raw data from Feature Extraction software was subsequently processed on GeneSpring GX 11.2 (Agilent Technologies). All the .txt raw data files are processed in the same Project, and the software creates a unique txt result file which contains the processed information for all samples (sample data file). That file contains the summarized data per biological feature and per sample. Experiment Part 1: Experiment type: Agilent Single Color Workflow: Advanced workflow 1. Transform all Feature Extraction flags to Absent/Marginal/Present (Compromised/Not detected/Detected in the file) calls, as follows: – Feature is NOT Positive and significant: ABSENT (Compromised in the file) – Feature is NOT uniform: ABSENT (Compromised in the file) – Feature is NOT well above background: ABSENT (Compromised in the file) – Feature is Saturated: MARGINAL (Not detected) – Feature is Population outlier: ABSENT (Compromised in the file) 2. Threshold raw signals (lineal data) to 1 3. Summarization: calculate the geometric mean of replicated probes. The resulted summarized lineal data is the “Raw Signal” in result files. 4. Log base2 transformation. 5. Per chip or intra-chip normalization: quantile normalization. The objective is the elimination or correction for the systematic error due to technical variations (sample processing, hybridization and/or washing artifacts, cross-hybridization, scanner settings,… ), that could hide the true biological variation. 6. Mean centering. The mean is centered to 0: for each sample, to each gene value the mean value for all samples for that gene is subtracted. The objective is to make comparable the gene expression for all arrays in the project. The result is the “Normalized Signal” (quantile + mean centering) in result files. 7. Filter Probesets based on Present/Absent calls: Use flag spot information in data files. Entities retained in which 80 % of samples in 1 out of any 6 conditions (3d Control, 3d Ionic, 3d Nano, 3w Control, 3w Ionic, 3w Nano) have Present or Marginal Flag.
Acute toxicity, bioaccumulation and effects of dietary transfer of silver from brine shrimps exposed to PVP/PEI-coated silver nanoparticles to zebrafish
