Cells were exposed for 6h or 24h at 37°C (95% RH, 5% CO2) to Ag-20, Ag-30, Ag-60, or Ag-110 AgNPs at a concentration of 25 µg/ml, or to AgNO3 at a concentration of 1.5 µg/ml (n=3)
Growth protocol
Both cell types were seeded at a density of ~40,000 ells/cm2 in a 6-well plate, and grown towards a confluency of ~60%.
Extracted molecule
total RNA
Extraction protocol
cells were washed to remove remaining nanoparticles, resuspended in 600 µl RLT buffer (RNeasy, Qiagen, Venlo, the Netherlands) containing 1% β-mercapto ethanol (Sigma). The lysate was further purified using the Qiashredder, followed by the RNeasy Mini Kit, including DNase treatment (Qiagen, Venlo, The Netherlands). RNA concentration and purity was determined using the Nanodrop ND1000 spectrophotometer (Isogen, De Meern, The Netherlands) and the Shimadzu MultiNA (Tokyo, Japan) and Agilent Bioanalyzer was used to analyze the quality and integrity of the RNA samples.
Label
biotin
Label protocol
Biotin (biotin-16-UTP) labeled cRNA was amplified from the total RNA using the Illumina TotalPrep-96 RNA Amplification Kit (Ambion, Life Technologies, Bleiswijk, the Netherlands).
Hybridization protocol
750 ng of each labeled cRNA sample was hybridized overnight at 58 °C onto the Illumina HumanHT-12 v4 Expression BeadChip.
Scan protocol
the BeadChip was washed and incubated with Streptavidin-Cy3 and the chips were scanned using the Illumina iScan array scanner (Illumina, San Diego, USA). Primary gene expression of the scanned BeadChip arrays was performed using Illumina’s Genomestudio v 2011.1 software with the default settings advised by Illumina.
Description
NA
Data processing
Normalization and analysis of the datasets was performed using MadMax. A variance Stabilization and Normalization (VSN) method was used to obtain expression values. After background subtraction the spot intensities were floored to 40, followed by 2 log mean centering and calculation of 2 log rations of treatments versus the average of the control samples.