treatment: Non radiated cell line id: 14663 genome/variation: BRCA1+/- brca1 mutation type: c.4327C>T Nonsense
Treatment protocol
DNA damage was induced through exposure to 2 Gy ionizing radiation (IR), delivered by a 137Cs Victoreen Electrometer (Atomic Energy of Canada, Mississauga, ON) at a dose rate of 0.52Gy/min. Following treatment, the cells were allowed to recover for a period of 6 hr at 37°C in 5% CO2 atmosphere prior to extraction of total RNA.
Growth protocol
Cells were cultured in RPMI-1640 media (Sigma Aldrich, Oakville, ON) supplemented with non-heat inactivated 15% fetal bovine serum (FBS) (Sigma Aldrich). All cell culture was carried out in 25cm2 flasks (Corning, Nepean, ON) at 37°C in 5% CO2 atmosphere. Cells were split in a 2:1 ratio until the desired cell number of 650,000 cells/ml was reached.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL® Reagent following the manufacturer’s recommendations (Invitrogen, Burlington,ON). RNA was purified using the RNeasy MinElute Cleanup Kit (Qiagen, Mississauga, ON). RNA quality was assessed by Agilent 2100 Bioanalyzer (Version B.02.02).
Label
Cy3
Label protocol
RNA with an integrity number of at least 7 was amplified and labeled using the Agilent Low RNA Input Linear Amplification kit (Agilent, Santa Clara, CA). Labeling reactions were performed with 250 ng total RNA, along with the Agilent Spike-in RNA mix, using Cy3-CTP and Cy5-CTP for control (-IR) and experimental (+IR) RNA, respectively (Perkin Elmer, MA, USA). Amplified RNA was quantified using the NanoDrop ND-1000 (NanoDrop Technologies, DE, USA) and the concentration of cRNA and the specific dye activity were calculated. Samples with a specific dye activity greater than 8 pmol/l were selected for hybridization to arrays.
DNA damage was induced through exposure to 2 Gy ionizing radiation (IR), delivered by a 137Cs Victoreen Electrometer (Atomic Energy of Canada, Mississauga, ON) at a dose rate of 0.52Gy/min. Following treatment, the cells were allowed to recover for a period of 6 hr at 37°C in 5% CO2 atmosphere prior to extraction of total RNA.
Growth protocol
Cells were cultured in RPMI-1640 media (Sigma Aldrich, Oakville, ON) supplemented with non-heat inactivated 15% fetal bovine serum (FBS) (Sigma Aldrich). All cell culture was carried out in 25cm2 flasks (Corning, Nepean, ON) at 37°C in 5% CO2 atmosphere. Cells were split in a 2:1 ratio until the desired cell number of 650,000 cells/ml was reached.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL® Reagent following the manufacturer’s recommendations (Invitrogen, Burlington,ON). RNA was purified using the RNeasy MinElute Cleanup Kit (Qiagen, Mississauga, ON). RNA quality was assessed by Agilent 2100 Bioanalyzer (Version B.02.02).
Label
Cy5
Label protocol
RNA with an integrity number of at least 7 was amplified and labeled using the Agilent Low RNA Input Linear Amplification kit (Agilent, Santa Clara, CA). Labeling reactions were performed with 250 ng total RNA, along with the Agilent Spike-in RNA mix, using Cy3-CTP and Cy5-CTP for control (-IR) and experimental (+IR) RNA, respectively (Perkin Elmer, MA, USA). Amplified RNA was quantified using the NanoDrop ND-1000 (NanoDrop Technologies, DE, USA) and the concentration of cRNA and the specific dye activity were calculated. Samples with a specific dye activity greater than 8 pmol/l were selected for hybridization to arrays.
Hybridization protocol
825ng Cy3 labeled control (-IR) and 825 Cy5 labeled experimental samples were mixed and hybridized to to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed with gene expression wash buffer 1 and 2.
Scan protocol
Image acquisition was done using an Agilent Microarray Scanner, Model G2565BA. Imaging analysis was done using Agilent Feature Extraction software v9.1.
Description
test set US45102857_251485013889_S01_GE2-v5_95_Feb07_1_2.txt
Data processing
Although the experiment was done to allow for analysis of radiation-dependent effects (Cy5/Cy3 ratio), the reported results are from unperturbed cells (Cy3 one color data only). The raw data is normalized by mean, per chip, using Excel. A total of 43,338 features were used in the analysis. A set of 38 features had gene label values that could not be interpreted by the analysis software, and were eliminated prior to analysis. Each sample was corrected for overall intensity by subtraction of the mean (per array) value from the value for each feature.
