Approximately 500,000 cells were seeded in 25 cm2 flasks and incubated for 24 hours. Medium was then exchanged for either fresh medium or medium prepared with 2,5 µM [Au(SCN)(PEt3)] and incubated for 24 hours prior to radiation treatment. Cells were further incubated for 24 hours immediately after irradiation and then medium was exchanged. For the purpose of gene expression analysis, cells were harvested 96 hours after subjection to ionizing radiation and stored in RNAlater (Qiagen, Valencia, CA) at -70 °C prior to RNA purification.
Growth protocol
Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C and 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from cells stored in RNA later using Rneasy (QIAGEN, Valencia, CA, USA)
Label
biotin
Label protocol
Total RNA were amplified and converted to cDNA and labeled using the Whole Transcript (WT) Sense Target Labeling Protocol
Hybridization protocol
From non-degraded high-quality total RNA we prepared and hybridized biotinylated complementary ssDNA to Rat Gene 1.0 ST Arrays, and then washed, stained and scanned slides using standardized protocols (Affymetrix Inc., Santa Clara, CA).
Scan protocol
We used standardized Affymetrix protocols (Affymetrix Inc., Santa Clara, CA)
Description
MS07
Data processing
Preprocessing was performed in Affymetrix Expression Console (v. 1.1) using the following methods: Summarization: PLIER, Background Correction: PM-GCBG, Normalization: Global Median
