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Sample GSM328755 Query DataSets for GSM328755
Status Public on Apr 27, 2009
Title PC-3 cells, passage 16-17, grown on Plastic, replicate 2
Sample type RNA
 
Source name PC-3 cells, passage 16-17, from ATCC
Organism Homo sapiens
Characteristics PC-3 human prostate cancer cell line, passage 16-17
Biomaterial provider ATCC (Manassas, VA)
Treatment protocol No specific treatment
Growth protocol PC-3 cells were maintained in phenol red-positive F-12 nutrient mixture (Invitrogen) containing 7% FBS and 100 units/ml penicillin-100 µg/ml streptomycin (25). PC-3 cells were passaged twice a week. PC-3 cells were used between passages 16 and 19.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated from prostate LNCaP and PC-3 cells for probe preparation and microarray hybridization. Briefly, 5x105 prostate cells were seeded in 60 mm tissue culture plates and cultured for 48 hours in their growth media. Total RNA was isolated from 4 consecutive passages from each cell line using RNeasy® Mini total RNA isolation kit following the manufacturer’s recommendations (Qiagen, Valencia, CA). The concentration of the isolated total RNA was determined with a Nanodrop scanning spectrophotometer, and then qualitatively assessed for their integrity using the ratio of 28:18s rRNA by a capillary gel electrophoresis system (Agilent 2100 Bioanalyzer, Agilent Technologies, Santa Clara CA).
Label Cy3
Label protocol A total of 250 ng of total RNA from each passage and from each cell line was labeled using the Illumina Total Prep RNA Amplification Kit following manufacturer’s directions (Ambion, Austin. TX). Briefly, cDNA was reverse transcribed from the total RNA after priming with T7-oligo-dT, and cRNA was synthesized in vitro from the T7 promoter with the incorporation of biotinylated UTP.
 
Hybridization protocol Biotinylated cRNA was hybridized overnight to Illumina human Ref-8 version 3 BeadChips containing probes for a total of 24,526 transcripts.
Scan protocol Microarray chips were washed to high stringency and labeled with streptavidin -Cy3 (Amersham Biosciences, Piscataway, NJ) prior to scanning on an Illumina BeadArray Reader.
Description No other descriptions
Data processing The data were first normalized based on normal distribution of low-level expressed genes using the previously described procedure (Dozmorov, Knowlton et al. 2004; Knowlton, Dozmorov et al. 2004). Normalization was performed for each array by first plotting a frequency histogram of the raw expression data for all genes. The histograms showed a right-skewed unimodal distribution curve with the mode around zero. Normal distribution curve representing variability of the data around zero was then fitted around the mode, mirroring Gaussian profile of the left part of the histogram. Two parameters [mean and standard deviation (SD)] were defined for this distribution; and the data were normalized to the SD of the noise after subtraction of the zero point. The arrays were than adjusted to each other by robust linear regression (Hua, Balagurunathan et al. 2006; Gusnanto, Calza et al. 2007).Genes with expression value less than 3.0 were considered to be not expressed. This is equivalent of setting a threshold at 3 SD above noise level, and each experiment accounted approximately 10,000 genes expressed below noise.
To identify differentially expressed genes between two experimental groups, we used associative analysis as described by Dozmorov et al. (Dozmorov and Centola 2003). Briefly, in pooled microarray datasets, a reference group of genes expressed above background with low variability of expression identified by F-test was identified. A t-test termed "associative t-test" was applied to test if a given gene belongs to or differs from this group. The associative t-test is a standard t-test applied to the comparison of expression variability. Due to a large number of genes that are differentially expressed between LNCaP and PC3 cells, a very stringent criterion was applied to identify genes with the most dramatic differences. Only genes expressed below noise level in one group and greater than 50 SD above noise level in another group were selected for further analysis. For genes that were expressed in both cell lines, genes that have greater than 10-fold differences in expression levels and expressed greater than 100 SD above noise level in at least in one cell line were also selected. These genes can be considered to be “beacons” that point to pathways or gene networks that are altered.
Dozmorov, I. and M. Centola (2003). "An associative analysis of gene expression array data." Bioinformatics 19(2): 204-211.
Dozmorov, I., N. Knowlton, et al. (2004). "Statistical monitoring of weak spots for improvement of normalization and ratio estimates in microarrays." BMC Bioinformatics 5(1): 53.
Gusnanto, A., S. Calza, et al. (2007). "Identification of differentially expressed genes and false discovery rate in microarray studies." Curr Opin Lipidol 18(2): 187-93.
Hua, J., Y. Balagurunathan, et al. (2006). "Normalization benefits microarray-based classification." EURASIP J Bioinform Syst Biol: 43056.
Knowlton, N., I. M. Dozmorov, et al. (2004). "Microarray Data Analysis Toolbox (MDAT): for normalization, adjustment and analysis of gene expression data." Bioinformatics 20(18): 3687-90.
 
Submission date Oct 08, 2008
Last update date Apr 27, 2009
Contact name Hsueh-Kung Lin
E-mail(s) hk-lin@ouhsc.edu
Phone 405-271-6900
Fax 405-271-3118
Organization name University of Oklahoma Health Sciences Center
Department Urology
Street address Room 462, 800 Research Parkway
City Oklahoma City
State/province OK
ZIP/Postal code 73104
Country USA
 
Platform ID GPL6883
Series (1)
GSE13131 Gene expression profiling of LNCaP and PC-3 prostate cancer cell lines

Data table header descriptions
ID_REF
SIGNAL_RAW Raw signal from BeadStudio
VALUE Log10 normalized to background, and adjusted across arrays by linear regression

Data table
ID_REF SIGNAL_RAW VALUE
ILMN_1809034 169.0696 1.816369399
ILMN_1660305 217.7399 1.929159226
ILMN_1762337 3.875936 0.20998707
ILMN_2055271 7.061933 0.44172717
ILMN_1814316 -3.813497 -4.782490579
ILMN_2359168 -3.709102 -4.782490579
ILMN_1731507 -1.347055 -4.782490579
ILMN_1787689 7.516427 0.466817488
ILMN_1745607 -6.387122 -4.782490579
ILMN_2136495 -1.803239 -4.782490579
ILMN_1668111 -4.252728 -4.782490579
ILMN_2295559 -1.976089 -4.782490579
ILMN_1735045 13.16053 0.698238764
ILMN_1680754 3.521596 0.174886518
ILMN_2375184 -0.5874797 -1.129291131
ILMN_1659452 4.095208 0.230405618
ILMN_1767388 -0.07803642 -0.614381994
ILMN_1675204 -4.327395 -4.782490579
ILMN_1673870 -0.9234763 -4.782490579
ILMN_1755321 48.64285 1.263908056

Total number of rows: 24526

Table truncated, full table size 822 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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