treatment: Alpha dose (gy): 1.5 time point (h) 1=24h 2=72h: 1 cell type: HLF
Treatment protocol
The cells were exposed to α-particle radiation at doses ranging from 0.0 (control) to 1.5 Gy, using 241Americium (241Am) electroplated discs (Eckert and Ziegler Isotope Products Ltd, Valencia, CA, USA) having an activity level of 66.0 kBq ± 3% (dose rate of 0.98 ± 0.01 Gy/h, LET of 127.4±0.4 keV/m).
Growth protocol
Cells were maintained in a humidified incubator (37oC, 5% CO2/95% air) in 75 cm2 tissue culture flasks (Costar, Cambridge, MA, USA). The cells were grown to confluence for 2-3 days in Royal Park Medical Institute-1640 (RPMI-1640) (Invitrogen Canada, Burlington, ON Canada) medium, containing 10% fetal bovine serum (FBS) (Sigma-Aldrich Canada, Oakville, ON, Canada). A total of 1.0x106 cells were seeded into 5 mL of culture media containing 100 units/mL of penicillin and 100 µg/mL of streptomycin (Invitrogen Canada Inc.).
Extracted molecule
total RNA
Extraction protocol
The pelleted cells were resuspended in 350 µL of Buffer RLT containing 1% β-Mercaptoethanol (Qiagen’s RNeasy Mini kit; Qiagen Inc, Mississauga, ON) then frozen at -80oC until processed. Frozen THP-1 cells were thawed on ice and mixed well by pipetting. The lysate was transferred directly onto a QIAshredder spin column (Qiagen Inc), placed in a 2 mL collection tube and centrifuged for 2 min at ~12,000 g. A volume of 350 µL of 70% ethanol was added. Total RNA was then extracted using the RNeasy Mini kit according to the manufacturer’s instructions (Qiagen Inc), with the addition of Qiagen’s On-Column RNase-free DNase (Qiagen Inc) to eliminate any remaining DNA contamination. All total RNA sample concentrations and RNA quality were determined using both an Agilent 2100 Bioanalyzer and RNA Nanochips (Agilent Technologies Canada Inc., Mississauga, ON) and spectrophotometrically using an Ultrospec 2100 (Fisher Scientific) (OD ratio of A260:A280). All extracted RNA samples were determined to be of good quality (RNA Integrity Number=10) with minimal degradation and stored at -80oC until further analysis. The pelleted cells were resuspended in 350 µL of Buffer RLT containing 1% β-Mercaptoethanol (Qiagen’s RNeasy Mini kit; Qiagen Inc, Mississauga, ON) then frozen at -80oC until processed. Frozen THP-1 cells were thawed on ice and mixed well by pipetting. The lysate was transferred directly onto a QIAshredder spin column (Qiagen Inc), placed in a 2 mL collection tube and centrifuged for 2 min at ~12,000 g. A volume of 350 µL of 70% ethanol was added. Total RNA was then extracted using the RNeasy Mini kit according to the manufacturer’s instructions (Qiagen Inc), with the addition of Qiagen’s On-Column RNase-free DNase (Qiagen Inc) to eliminate any remaining DNA contamination. All total RNA sample concentrations and RNA quality were determined using both an Agilent 2100 Bioanalyzer and RNA Nanochips (Agilent Technologies Canada Inc., Mississauga, ON) and spectrophotometrically using an Ultrospec 2100 (Fisher Scientific) (OD ratio of A260:A280). All extracted RNA samples were determined to be of good quality (RNA Integrity Number=10) with minimal degradation and stored at -80oC until further analysis.
Label
biotin
Label protocol
cRNA was labeled using biotin following protocols in Genome Expression Profiling Assay Guide (11317302 Rev. A).
Hybridization protocol
Samples were hybridized on Illumina human-12 v3 RNA BeadChips.
Scan protocol
BeadChips were imaged and quantified with the Illumina iScan scanner.
Description
24h
Data processing
Data pre-processing was carried out within GenomeStudio v2010.2.2. The data was median normalized (50th percentile) where the intensities were averaged per probe/gene. Normalization of dataset was conducted in GeneSpring (version GX 11.5).
