SUMMARY: Response to radiation in prostate adenocarcinoma. LNCaP C4-2 cells, polyA+ mRNA extracted, fractionated: harvested 6 hr after the fourth of four sequential daily 2.5 Sv radiation doses, Affymetrix HGU-95E GeneChip(R). DESIGN: The experiment design was multifold, consisting of a time course experiment on LNCaP C4-2 human prostate adenocarcinoma cells following irradiation to a dose of 10 Sv from a Cesium-137 mixed gamma and beta source, with two additional samples, one exposed to radiation in four fractions instead of all at once, and one containing the parent LNCaP cell line. PolyA+ RNA was extracted, processed and hybridized to the Affymetrix (Santa Clara, CA, www.affymetrix.com) HGU-95 (Av1-E) chip sets, 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 parent LNCaP cell lines were obtained from ATCC (Manassas, VA). The derived LNCaP 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: The sample was irradiated to a dose of 10 Sv in four equal fractions of 2.5 Sv given over four consecutive days; the sample was harvested 6 hr after the final dose. The radiation source was a J.L. Shepherd and Associates (San Fernando, CA) Irradiator containing a Cesium 137 source. The dose was delivered at a rate of 2.8 Sv/min. Cesium 137 is a mixed beta/gamma emmitter; the gamma ray emitted has an energy of 661.657 keV. For details see, for example, www.bnl.gov/ton. EXTRACTION: PolyA+ mRNA was prepared from total RNA by using QIAGEN Oligotex kits. PROCESSING: Sample processing proceeded as recommended in the Affymetrix GeneChip(R) Expression Analysis Manual. Double-stranded cDNA was prepared from the PolyA+ mRNA 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 HGU-95E 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, FlexGE: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:832, Count:32 Corner- Avg:35095, Count:32 Background Avg:414.13, Stdev:5.46, Max:426.4, Min:398.7 Noise Avg:12.35, Stdev:0.40, Max:13.9, Min:11.5 RawQ 17.92 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 4.387765 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 (A-E) 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
