SUMMARY: Kinetic response to radiation in prostate adenocarcinoma. LNCaP C4-2 cells, total RNA extracted, harvested 20 hr after irradiation to a dose of 10 Sv, Affymetrix HG-U95Av2 GeneChip(R). DESIGN: The experiment was a time course experiment on LNCaP C4-2 human prostate adenocarcinoma cells following irradiation to a dose of 10 Sv from a Cesium-137 gamma source. Total RNA was extracted from cells at 1, 2, 4, 6, 8, 12, 16, 20 and 24 hours after irradiation. The untreated control sample, labeled 0, was collected concurrently with the cells extracted at 24 hr. After extraction, the samples were processed and hybridized to the Affymetrix (Santa Clara, CA, www.affymetrix.com) HG-U95Av2 chip, and then washed, stained and scanned according to Affymetrix's protocols contained in the GeneChip(R) Expression Analysis Manual. Transcript abundance data from the scans was processed initially with Affymetrix' Microarray Suite 5(R), then by Silicon Genetics' (Redwood City, CA, www.silicongenetics.com) GeneSpring(R). BIOLOGICAL SAMPLE: Cultured LNCaP C4-2 human prostate adenocarcinoma cells. The derived LNCaP C4-2 cell lines are described in Thalmann, GN, et.al., Cancer Res., 54:2577-2581, 1994, PMID: 8168083. (See also Ray, S and Almasan, A, Cancer Res, 63:4713-4723, 2003, PMID: 1290765.) The C4-2 derivative of the LNCaP cell line is androgen independent. IRRADIATION: Harvested at specified intervals after irradiation to a dose of 10 Sv from a J.L. Shepherd and Associates (San Fernando, CA) Mark 1 Irradiator containing a Cesium 137 source. The dose was delivered at a rate of 2.8 Sv/min. Cesium 137 emits a gamma ray with an energy of 661.657 keV. For details see, for example, www.bnl.gov/ton. EXTRACTION: Total RNA was extracted from cells using the Trizol reagent (GIBCO BRL). PROCESSING: Sample processing proceeded as recommended in the Affymetrix GeneChip(R) Expression Analysis Manual. Double-stranded cDNA was prepared from total RNA using the GIBCO BRL SuperScript Choice system. An in vitro transcription reaction was used to produce biotin-labeled cRNA using the Enzo BioArray High Yield RNA Transcript Labeling Kit. The sample was then purified using the QIAGEN RNA MiniKit and then fragmented. A hybridization mixture was prepared including the fragmented cRNA, the Affymetrix recommended control cocktail and herring sperm DNA. MICROARRAY: Affymetrix HG-U95Av2 GeneChip(R). HYBRIDIZATION: Affymetrix Hybridization Oven 640, Time: 16 hr, Temp: 45 C, Carousel Rotation Speed: 60 rpm. WASHING AND STAINING: Affymetrix Fluidics Station 400, EukGE:WS2v3 Protocol. SCANNING: Affymetrix Agilent 2500 Scanner, Pixel Size: 3 microns, Filter: 570 nm, Scans: 2. Original calibration. The source of the data recorded below is the scan before the final antibody stain. (See the Affymetrix technical note, GeneArray(R) Scanner Update, Part 700575 Revision 2.) DEVICE SPECIFIC ANALYSIS: Affymetrix Microarray Suite 5.0 Algorithm: Statistical Corner+ Avg:491, Count:32 Corner- Avg:33215, Count:32 Background Avg:320.97, Stdev:2.74, Max:327.9, Min:314.7 Noise Avg:9.88, Stdev:0.28, Max:11.0, Min:8.9 RawQ 13.38 Alpha1 0.04 Alpha2 0.06 Tau 0.015 Gamma1H 0.0025 Gamma1L 0.0025 Gamma2H 0.003 Gamma2L 0.003 Perturbation 1.1 TGT 300 NF 1.000000 SF 1.501031 SFGene All DEVICE INDEPENDENT ANALYSIS: Silicon Genetics' GeneSpring(R) version 6, build 1333. The Microarray Suite 5 output data was further processed with GeneSpring(R). Each chip was normalized separately. Each measurement on the chip was divided by the 50th percentile of all measurements (the median) in that sample. The percentile was calculated with all normalized measurements above 0. Values below 0.0 were set to 0.0. Keywords = Human Keywords = Prostate Keywords = Cancer Keywords = Adenocarcinoma Keywords = Ionizing Radiation Keywords = Microarray
