cell line: 16lu treatment: MWCNT w/o IL-13 time: 4 h
Growth protocol
A separate, frozen stock was used for each experiment. Individual stocks were thawed and expanded by growth in Dulbecco’s Modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotic-antimycotic (Invitrogen, Carlsbad, CA). Cells were passaged twice in 175 cm2 flasks, liberated with trypsin-EDTA, and seeded in 100 cm2 plates with 0.5-1x105 cells per plate. Cells were grown to confluence and culture medium replaced with a serum-free defined medium (SFDM) comprised of Ham’s F12, 0.25% BSA, 1 g/L insulin, 0.55 g/L transferrin, 0.67 mg/L sodium selenite and antibiotic-antimycotic 24 h prior to treatment.
Extracted molecule
total RNA
Extraction protocol
A two-phase purification method was used to isolate mRNA with reduced nanoparticle contamination. RNALater® was removed by centrifugation and washing with DPBS. RNA was isolated by adding 1 ml RNA STAT-60 reagent (Sigma-Alrich, St. Louis, MO) to microfuge tubes for 5-10 min, extraction with 0.2 ml chloroform, and isolation of the aqueous phase. The majority of remaining MWCNT and CBNP were effectively trapped in the phenol/chloroform phase or were co-precipitated with denatured proteins. The clear aqueous phase was isolated and RNA precipitated with isopropanol and washed with 70% ethanol. The resulting pellet was resuspended in 100 µl RNAse-free water and 350 µl of buffer RLT added prior to transferring to an RNEasy spin column. Purification then proceeded according to the manufacturer’s instructions for the RNEasy Mini Kit and included an on-column DNAse I digestion (Qiagen, Valencia, CA). RNA quality verified spectrophotometrically on a Nano Drop 1000 (Nano Drop, Inc., Wilmonton, DE) and by gel electrophoresis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Biotin
Label protocol
stranded cDNA was synthesized from RNA using an oligo-dT24-T7. Biotinylated cRNA was synthesized from an aliquot of the cDNA template using the T7 RNA Transcript Labeling Kit (ENZO Diagnostics, Farmingdale NY).
Hybridization protocol
The labeled cRNA was fragmented, hybridized to Affymetrix Human Genome U133A 2.0 arrays (Affymetrix, Santa Clara, CA), and stained using phycoerythreinconjugated streptavidin (Molecular Probes, Eugene, OR).
Scan protocol
Affymetrix Standard Protocol
Description
_6__24_MW_4_h___HG_U133A_2_JCBLR
Data processing
Data analysis: Affymetrix Cel files produced from the expression experiment were analyzed using JMP Genomics 4.1 software from SAS Institute. Data normalization was performed using a Standard Normalization implemented by the Data Standardize AP in JMP Genomics. Expression values were standardized to the mean with a scale equivalent to the standard deviation.
