irradiated (4gy): NO radioresistant: Radioresistant overexpression beta cat: NA
Treatment protocol
SW1463 cells were repeatedly irradiated with 2 Gy of X-rays for 34 cycles, equaling to a cumulative dose of 68 Gy. Specifically, cells were seeded into six-well plates, irradiated the next day, and cultured in fresh medium. At 70-80% confluence, they were exposed to the next cycle of irradiation.
Growth protocol
Cell lines, SW1463 directly from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in their recommended media, supplemented with 10% fetal bovine serum and 2 mM l-glutamine, and discarded no later than 15 passages. Periodically, mycoplasma contamination was determined using the MycoAlert® Mycoplasma Detection Kit.
Extracted molecule
total RNA
Extraction protocol
For each condition (i.e., no irradiation or 6 hours after exposure to 4 Gy of X-rays), total RNA was isolated from logarithmic growing cells at three different passages (passages four to six after thawing) using the RNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions, including the optional on-column DNase digestion. Quantity and purity of isolated RNA was assessed using a NanoDrop ND-1000 spectrophotometer (ThermoFischer, Waltham, MA); RNA integrity was assessed by Agilent 2100 Bioanalyzer electrophoresis (Agilent Technologies, Palo Alto, CA). Only samples with an RNA integrity number >9.5 were considered for additional experiments.
Label
Cy3
Label protocol
200 ng of total RNA was reverse transcribed, amplified, and labeled with Cy3 using the Low RNA Input Linear Amplification Kit PLUS (Agilent Technologies)
Hybridization protocol
1.65 μg of labeled cRNA was fragmentized and hybridized overnight to a 4 x 44k gene expression microarray (Agilent Technologies)
Scan protocol
Arrays were scanned on an Agilent DNA microarray scanner G2505B (Agilent Technologies) at 5-micron resolution.
Data processing
Raw data were log2 transformed and normalized to 75 percentile according to Agilent protocol. Probes with intensity no less than 100 in at least one sample were kept for analysis.
