organ: lung treatment: (0.8 cm2/cm2) of NM403 for 4 h
Treatment protocol
Cells were seeded in 25 cm2 flasks at a density of 3 x 105 cells / mL for 48 h.
Growth protocol
NR8383 cell line were established from normal rat by lung lavage and was obtained from American Type Culture Collection (ATCC® CRL-2192™, Manassas, VA, USA). Cells, with a 50% adherent and 50% floating cell phenotype, were grown in DMEM medium supplemented with 15 % of heat-inactivated fetal bovine serum, 2 mM of L-glutamine, as well as 100 U/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of amphotericin B (all from Sigma Aldrich). Cells were incubated at 37°C in an atmosphere composed of 95 % air, 5 % CO2 and medium are changed every 2-3 days.
Extracted molecule
total RNA
Extraction protocol
The quality of the RNA extracted by RNA-Solv Reagent (Omega Bio-tek, USA) was determined by spectrophotometry (A260nm/A280nm, A260nm/A230nm > 1.8, BioSpec-nano, Shimadzu) and by capillary electrophoresis using RNA 6000 Nano® (RIN > 7.5, Agilent 2100 Bioanalyzer, Santa Clara, CA, USA).
Label
CTP-Cy3
Label protocol
CTP-cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color Microarray-Based Gene Expression Analysis (Agilent) according to manufacturer's instructions, followed by chromatographic purification (RNeasy Mini kit, QIAGEN). Dye incorporation and cRNA yield and quality were checked by spectrophotometry (specific activity > 10 pmol Cy3 / µg cRNA, BioSpec-nano, Shimadzu) and by electrophoresis (Agilent 2100 Bioanalyzer).
Hybridization protocol
Six hundred ng of CTP-Cy3-labelled cRNA were fragmented at 60°C for 30 min in a reaction volume of 25 µL following the manufacturer’s instructions. 25 µL of 2x Agilent hybridization buffer were added to the fragmentation mixture and cRNA were hybridized to SurePrint G3 Rat GE v2 8x60K (Agilent) for 17 h at 65°C in a rotating hybridization oven. After hybridization, microarrays were washed 1 min at room temperature with GE Wash Buffer 1TM and 1 min with 37°C GE Wash buffer 2TM (Agilent).
Scan protocol
Slides were scanned immediately after washing using Agilent DNA Microarray Scanner (G2505C) by setting: (i) one color scan channel for 8x60k array slides, (ii) scan area of 61x21.6 mm, (iii) scan resolution of 3 µm, (iv) dye channel to Green, (v) Tiff file dynamic range of 20 bits and (vi) Green PMT to 100 %.
Description
Gene expression in NR8383 cells after 20 passages
Data processing
The scanned images were analyzed by Feature Extraction version 11.0.1.1 (Agilent) using default parameters (protocol GE1_1100_Jul11 and Grid: 074036_D_F_20150914) to subtract background and to obtain spatially detrended processed signal intensities. Features flagged in Feature Extraction as Non-uniform outliers Feature were excluded.
