8 protocols
The functions used for normalization were implemented in Matlab and in Bioconductor package diffGeneAnalysis. Raw expression values were first normalized based on normal distribution of genes with low expression levels as previously described [25, 26]. Briefly, 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 random variability of the genes with low expression values 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. The expression values then were normalized to the SD of the normal distribution after subtraction of the mean and log10-transformed. The arrays were than adjusted to each other by robust linear regression [27, 28]. Genes with expression value less than 3.0 (or 0.477 in log10 scale) were considered to be not expressed; this is equivalent of setting a threshold at 3 SD above noise level. 25. Dozmorov I, Knowlton N, Tang Y, Centola M: Statistical monitoring of weak spots for improvement of normalization and ratio estimates in microarrays. BMC Bioinformatics 2004, 5:53. 26. Knowlton N, Dozmorov IM, Centola M: Microarray Data Analysis Toolbox (MDAT): for normalization, adjustment and analysis of gene expression data. Bioinformatics 2004, 20:3687-3690. 27. Hua J, Balagurunathan Y, Chen Y, Lowey J, Bittner ML, Xiong Z, Suh E, Dougherty ER: Normalization benefits microarray-based classification. EURASIP journal on bioinformatics & systems biology 2006:43056. 28. Gusnanto A, Calza S, Pawitan Y: Identification of differentially expressed genes and false discovery rate in microarray studies. Curr Opin Lipidol 2007, 18:187-193.
Biotinylated cRNA was hybridized to Illumina HumanWG 6 v2 BeadChips containing a total of 48,702 probes.
AKR1C3 expression construct (pcDNA3-AKR1C3) was established as previously described [24]. Transfection of PC-3 cells with either pcDNA3 empty vector or pcDNA3-AKR1C3 construct was achieved using Lipofectamine 2000; and stable transfectants were established in the presence of G418 selection (600 μg/ml). Multiple independent clones were isolated and expanded in the complete growth medium containing G418. PC-3 stable transfectants were designated as PC3-mock or PC3-AKR1C3 for cells stably transfected with pcDNA3 or pcDNA3-AKR1C3 plasmids, respectively. Three independent PC3-AKR1C3 clones confirmed with elevated AKR1C3 expression as compared to PC3-mock transfectants were used for bioinformatics and biological analyses [24]. 24. Wang S, Yang Q, Fung KM, Lin HK: AKR1C2 and AKR1C3 mediated prostaglandin D2 metabolism augments the PI3K/Akt proliferative signaling pathway in human prostate cancer cells. Mol Cell Endocrinol 2008, 289:60-66.
A total of 250 ng of total RNA from each clone was labeled using the Illumina Total Prep RNA Amplification kit (Ambion, Austin. TX). cDNA was reverse transcribed from the total RNA after priming with T7-oligo(dT); and cRNA was synthesized from the T7 promoter in the presence of biotinylated UTP.
VALUE = log10-transformed
Microarray chips were labeled with streptavidin -Cy3 (Amersham Biosciences, Piscataway, NJ) and scanned on an Illumina BeadArray Reader.
Total RNA was isolated from 2 independent PC3-mock clones and 3 independent PC3-AKR1C3 clones for gene expression analysis. Briefly, following PC-3 transfectants (5x105) seeding and adherence, total RNA was isolated from these clones using RNeasy® Mini total RNA isolation kit (Qiagen, Valencia, CA). The concentration of the isolated total RNA was determined with a Nanodrop scanning spectrophotometer, and then qualitatively assessed using the ratio of 28:18s rRNA by Agilent 2100 Bioanalyzer capillary gel electrophoresis system (Agilent Technologies, Santa Clara CA).
PC-3 cells were maintained in a complete growth medium consisting of F-12 nutrient mixture supplemented with 7% FBS and 1% penicillin-streptomycin in a humidified cell incubator at 37 oC and 5% CO2.