cell line: NCI-H460 cell type: human lung large cell carcinoma cells treatment: nicotine timepoint: 2 hours
Treatment protocol
Nictoine (5 uM; Sigma®, Saint Louis, MO) or cigarette smoke extract (5 uM nicotine-equivalent) was added to the culture media, and after 2 hours, cells were irradiated with one dose of 6 Gy ionizing radiation (cesium-137 source), or cisplatin (1 uM; Sigma®) was added to the culture medium. All treatments were performed concurrently using a total of nine 6-well cuture plates. After another 2 or 24 hours, cells were harvested by scraping into the culture medium, spun down, washed in 10 ml phosphate-buffered saline, and collected as cell-pellets after another round of centrifugation at room temperature. The total 54 cell-pellets were stored frozen at -80 ºC until RNA extraction.
Growth protocol
3x10e5 NCI-H460 cells, at passage number 9 after procurement from ATCC® (Manassas, VA) and maintained in RPMI-1640 medium (Life Technologies®, Grand Island, NY) with 10% fetal bovine serum (Atlanta Biologicals®, Lawrenceville, GA) at 37 ºC under 5% carbon dioxide, were seeded per well of 6-well tissue culture dishes. After 24 hours, the culture medium was replaced with that without serum and cells were grown for another 24 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from -80 ºC-frozen cell-pellets using the Total RNA Purification kit (catalog# 37500, Norgen-Biotek®, Thorold, ON, Canada) following the kit manufacturer's guidelines. The kit uses silicon carbide spin-columns. On-column DNase I treatment was performed to reduce contaminating genomic DNA. RNA eluted from the columns was treated on-column with DNase I again as per the manufacturer's guidelines provided with the RNase-Free DNase I kit (Norgen-Biotek®). Absorbance at 260 nm and electrophoresis on Bioanalyzer™ 2100 (Agilent®, Austin, TX) was used to estimate RNA concentration and quality.
Label
Biotin
Label protocol
The WT Expression kit from Ambion® (Austin, TX) was used to generate sense-strand complementary DNA (cDNA) from 100 ng total RNA. The cDNA was fragmented and labeled with biotin as per protocols provided with the GeneChip™ WT Terminal Labeling and Hybridization kit (Affymetrix®). This work was performed as a commercial service by a shared genomic facility of the University at Buffalo, Buffalo, NY.
Hybridization protocol
Hybridization and washes were performed on an Affymetrix® GeneChip™ Fluidics Station 450 instrument using protocols provided by Affymetrix®. This work was performed as a commercial service by a shared genomic facility of the University at Buffalo, Buffalo, NY.
Scan protocol
Arrays were scanned on an Affymetrix® GeneChip™ Scanner 3000 instrument using protocols provided by Affymetrix®. This work was performed as a commercial service by a shared genomic facility of the University at Buffalo, Buffalo, NY.
Description
Sample 5 Biological replicate 2 of 3.
Data processing
Raw data for all 54 arrays (in .CEL files) was processed together using Affymetrix® Power Tools (version 1.15.0) on Mac OS X 10.6.8. Briefly, background signals were adjusted using the 'rma' method. Array data was then quantile normalized and median polish summarized. All arrays appeared to have yielded data of good quality as assessed using criteria suggested by Affymetrix®.
Effect of cigarette smoke extract, cisplatin, nicotine and/or ionizing radiation on gene expression in the NCI-H460 human lung large cell carcinoma cell line
Effect of cigarette smoke extract, cisplatin, nicotine and/or ionizing radiation on mRNA and microRNA expression in the NCI-H460 human lung large cell carcinoma cell line
