cell line: tumor cell line 9 (gammaA8) disease state: lung cancer
Growth protocol
Each cell line was passaged weekly and maintained in culture for up to 50 passages. Eleven transformed mouse cell lines from lung tumor of double c-Myc and c-Raf transgenic mice were used for this project (A2B1, A2C12, B3, βD5, βD10, c-Myc_c-Raf, γA3, γA7, γB8, γD1, γD12). The tumor cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, PAA, Pashing, Austria) supplemented with 10% fetal calf serum (FCS, FHG, Pashing, Austria), 2mM L-Glutamin, 100units/ml streptomycin and 100µg/ml penicillin in a humidified atmosphere at 37ºC containing 5% CO2 to maintain the medium pH at a value of 7,4. Alveolar epithelial cells (AECs) were used as control.
Extracted molecule
total RNA
Extraction protocol
RNA-extraction was performed with the RNeasy Mini Kit (RNeasy MidiKit Qiagen, Santa Clarita, CA, USA) according to the manufacturer’s instruction. A standard quality control of the total RNA was performed using the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA).
Label
biotin
Label protocol
Total RNA (5µg) was used to generate biotin-labeled cRNA (10 µg) by means of GeneChip® One-Cycle cDNA Synthesis Kit and GeneChip® IVT Labeling Kit (Affymetrix). Sample processing was performed exactly as described by the microarray manufacturer (Affymetrix). Quality control of cRNA was performed using a Bioanalyzer (Agilent 2001 Biosizing, Agilent Technologies).
Hybridization protocol
Following fragmentation, labeled cRNA of each sample was hybridized to Affymetrix GeneChip® Mouse Genome -U74v2 Arrays covering approximately 12,000 full-length mouse gene transcripts and stained according to the manufacturer's instructions.
Scan protocol
The arrays were scanned using the GeneChip® Scanner 3000.
Description
tumor cell line 9 Isolation of control and tumor cells: Alveolar epithelial type II cells and tumor cells were isolated as originally described by Richards et al. (1987) [129], with the following modifications: Briefly, 8-month-old transgenic mice were anesthetized by intraperitoneal injection of Ketamin® (100 mg/kg) and Rompun® (10 mg/kg). After tracheotomy the trachea was cannulated with a bulb head cannula. Following midline incision and removal of the rib cage, a Luer cannula was inserted via the vena cava cranialis into the right atrium. The pulmonary circulation was perfused with ice cold PBS using a peristaltic pump at a flow rate of 4 ml/min. The perfusate was removed by incision of the left atrium, and perfusion with the buffer was continued for 2–3 min until the lungs were completely free of blood. The lungs were explanted, placed in a 50 ml Falcon tube containing 30 ml ice cold PBS and transferred to a sterile flow cabinet. The lungs were then lavaged four times with 6 ml PBS, and subsequently 6 ml Trypsin–EDTA solution (0.25%) was instilled into the trachea. The lungs were incubated with trypsin at 37 °C for a total of 45 min. Every 15 min, 4 ml trypsin solution was pumped via the tracheal cannula.The trachea and main bronchi were removed, and the parenchymal lung tissue was dissected into 1- to 2-mm pieces in a petri dish containing 10 ml ice cold PBS with Ca2+. The lung tissue suspension was incubated for 10 min with 10 ml DNase solution at a concentration of 250 μg/ml DNase and then filtered through a nylon mesh (100 and 60 μm pore size). The alveolar type II pneumocytes were prepared by discontinuous percoll gradient centrifugation (heavy density 1.089 and low density 1.040). Following centrifugation for 30 min at 250g and 4 °C, the cell fraction at the interface between the heavy and low-density gradient was removed and mixed with DNase solution containing 50 μg/ml DNase. The cell suspension was again centrifuged at 140g at 4 °C for 6 min. The resulting cell pellet was then resuspended in DMEM supplemented with 10% FCS or 5% rat serum and incubated in a 25 cm2 tissue culture flask at 37 °C for 60 min. The non-adherent cells were removed and plated on collagen-coated 12-well plates. Approximately 2 × 106 cells were seeded per cm2 in 1 ml culture medium.
Data processing
Array data was normalized using scaling or per-chip normalization to adjust the total or average intensity of each array to be approximately the same. Microarray chips were analyzed by the GCOS (GeneChip Operating Software) from Affymetrix with the default settings except that the target signal was set to 250 and used to generate a microarray quality control and data report. First, we have evaluated and compared multiple data from each tumor cell line using statistical analysis with the Affymetrix Data Mining Tool (DMT). Since significant differences observed between the tumor cell lines we prepared additionally two replicates from tumor cell lines with most differences such as A2C12, cRaf_cMyc, A2B1, B3, gammaA7 and gammaB8 and conducted further data analysis. CEL files exported from GCOS were uploaded into ArrayTrack software (National Center for Toxicological Research, U.S. FDA, Jefferson, AR, USA (NCTR/FDA)).
