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11-01-06 MIAME Checklist for Normal Variation Paper Experiment Design: The goal of the experiment – one line maximum (e.g., the title from the related publication) Title: Genetic background influences murine prostate gene expression: Associations with cancer phenotypes in man. A brief description of the experiment (e.g., the abstract from the related publication): Cancer of the prostate is influenced both by genetic predisposition and environmental factors. The identification of genes capable of modulating cancer development has the potential to unravel disease heterogeneity and aid diagnostic and prevention strategies through improved understanding of geneenvironment interactions. To this end, mouse models have been developed to isolate the influences of individual genetic lesions in the context of consistent genotypes and environmental exposures. However, the extent of normal prostatic phenotypic variability dictated by a genetic background potentially capable of influencing the process of carcinogenesis has not been established. In this study we used microarray analysis to quantitate transcript abundance levels in the prostates of five commonly studied inbred mouse strains. We applied a multiclass response t-test to identify genes whose expression in each strain significantly differed from the other four strains. Approximately 13% (932 genes) exhibited significant differential expression (range 1.3 to 190fold) in one strain relative to other strains (FDR≤10%). The pattern of variability in transcript levels did not result from variations unique to a particular strain, but rather represented genetic variability across all five strains assessed. Expression differences were confirmed by qRT-PCR, or immunohistochemistry for several genes previously shown to influence cancer progression such as Psca, Mmp7, and Clusterin. Analyses of human prostate transcripts orthologous to variable murine prostate genes identified differences in gene expression in benign epithelium that correlated with the differentiation state of adjacent tumors. For example, the expression of apolipoprotein D, a gene known to enhance resistance to cell stress in Drosophila, was expressed at significantly greater levels in benign epithelium associated with high-grade versus low-grade cancers. These data support the concept that the cellular, tissue, and organismal context contribute to oncogenesis and suggest that a predisposition to a sequence of events leading to pathology may be determined prior to cancer initiation. Keywords, for example, deletion, gain, amplification, polymorphisms detection, strain determination (the use of MGED ontology terms is recommended): Keywords: mouse, prostate, transcript, variation, microarray, cancer, apolipoprotein D Experimental factors - the parameters or conditions tested, for example, disease state, strain, species isolate (the use of MGED ontology terms is recommended): Genetic variation Experimental design - relationships between samples, treatments, extracts, labeling, and arrays (e.g., a diagram or table) Experimental design. Prostates from twelve mice from each of five strains of Mus musculus: C57BL/6J, 129X1/SvJ, BALB/cAnNCrl, FVB/NJ and DBA/2NCrl, were resected and individual lobes were dissected: DP: dorsal prostate; LP: lateral prostate; VP: ventral prostate; AP: anterior prostate. Each experimental sample represents a pool of equal amounts of RNA for each prostatic lobe from 3 animals. Four independent experimental samples were created per strain: 12 mice divided into 4 pools of 3 mice each for a total of 4 microarray experiments per strain. Amplified RNA from each experimental sample was hybridized against a reference sample (created by combining equal amounts of RNA from all the samples from all strains) onto custom mouse prostate cDNA microarrays using alternate dye-labeling to account for dye-specific effects. Quality control steps taken (e.g., replicates or dye swaps): 1) 12 mice, 4 pools of 3 mice per strain for a total of 4 microarray experiments per strain 2) Half of the biological experimental samples per strain were labeled with Cy3 dye and the reference with Cy5 dye, and in the other half the labeling was inversed. Links to the publication, any supplemental websites or database accession numbers: http://www.mpedb.org/SUPPLEMENTARY/SUBMITTED/DBIANCHI Samples used, extract preparation and labelling: The origin of each biological sample (e.g., name of the organism, the provider of the sample) and its characteristics (e.g., gender, age, developmental stage, strain, or disease state). This information must not compromise the patients anonymity – legal and ethical requirements take the precedence. Table 1. Mouse strain Strain 129X1/SvJ C57BL/6J FVB/NJ DBA/2NCrl BALB/cAnNCrl Vendor Jackson Labs Jackson Labs Jackson Labs Charles River Charles River Stock number 691 664 1800 026 028 Sex MALE MALE MALE MALE MALE Age 7 weeks 7 weeks 7 weeks 7 weeks 7 weeks Table 2. Experimental samples # Sample pool ID Strain Lobe Age Pool Components 1 129-1 129X1/SvJ DP, LP, VP and AP 8-9 weeks 129X1/SvJ; mouse # 1, 5 and 12 2 129-2 129X1/SvJ DP, LP, VP and AP 8-9 weeks 129X1/SvJ; mouse # 2, 7 and 11 3 129-3 129X1/SvJ DP, LP, VP and AP 8-9 weeks 129X1/SvJ; mouse # 3, 6 and 9 4 129-4 129X1/SvJ DP, LP, VP and AP 8-9 weeks 129X1/SvJ; mouse # 4, 8 and 10 5 Balb-1 BALB/cAnNCrl DP, LP, VP and AP 8-9 weeks BALB/cAnNCrl; mouse # 1, 5 and 12 6 Balb-2 BALB/cAnNCrl DP, LP, VP and AP 8-9 weeks BALB/cAnNCrl; mouse # 2, 7 and 10 7 Balb-3 BALB/cAnNCrl DP, LP, VP and AP 8-9 weeks BALB/cAnNCrl; mouse # 3, 6 and 11 8 Balb-4 BALB/cAnNCrl DP, LP, VP and AP 8-9 weeks BALB/cAnNCrl; mouse # 4, 8 and 9 9 C57-1 C57BL/6J DP, LP, VP and AP 8-9 weeks C57BL/6J; mouse # 1, 6 and 11 10 C57-2 C57BL/6J DP, LP, VP and AP 8-9 weeks C57BL/6J; mouse # 2, 7 and 10 11 C57-3 C57BL/6J DP, LP, VP and AP 8-9 weeks C57BL/6J; mouse # 3, 4 and 9 12 C57-4 C57BL/6J DP, LP, VP and AP 8-9 weeks C57BL/6J; mouse # 5, 8 and 12 13 DBA-1 DBA/2NCrl DP, LP, VP and AP 8-9 weeks DBA/2NCrl; mouse # 1, 8 and 11 14 DBA-2 DBA/2NCrl DP, LP, VP and AP 8-9 weeks DBA/2NCrl; mouse # 2, 5 and 12 15 DBA-3 DBA/2NCrl DP, LP, VP and AP 8-9 weeks DBA/2NCrl; mouse # 3, 6 and 9 16 DBA-4 DBA/2NCrl DP, LP, VP and AP 8-9 weeks DBA/2NCrl; mouse # 4, 7 and 10 17 FVB-1 FVB/NJ DP, LP, VP and AP 8-9 weeks FVB/NJ; mouse # 1, 5 and 12 18 FVB-2 FVB/NJ DP, LP, VP and AP 8-9 weeks FVB/NJ; mouse # 2, 7 and 11 19 FVB-3 FVB/NJ DP, LP, VP and AP 8-9 weeks FVB/NJ; mouse # 3, 6 and 9 20 FVB-4 FVB/NJ DP, LP, VP and AP 8-9 weeks FVB/NJ; mouse # 4, 8 and 10 Manipulation of biological samples and protocols used (e.g., stress, drugs). NA Experimental factor value for each experimental factor, for each sample (e.g., ‘disease state = AML’ for a sample in a time course experiment). Strain differences Technical protocols for preparing the hybridization extract and labeling: Prostate tissues were homogenized using a Polytron and total RNA was isolated using the RNeasy extraction kit (Qiagen). Equal amounts of total RNA (0.7 µg) from each of the four pooled lobes/strain were combined to produce secondary pools, and amplified with the MessageAmpTM aRNA kit (Ambion). Amplified RNA from each secondary pool (four pools per strain) was hybridized separately to cDNA microarrays for a total of 4 independent biological replicates per strain (12 mice, 4 pools of 3 mice per strain for a total of 4 microarray experiments per strain; see Figure 1). A common reference sample was created by combining equal amounts of RNA from all the samples from all strains. The amplified RNA was used as template for cDNA probe synthesis followed by hybridization to a custom mouse prostate cDNA array (mPEDB array), comprised of ~8300 genes expressed in the developing and adult mouse prostate (http://www.mpedb.org/). The 20 microarrays used for the experiment were all from the same printing batch. The protocol used for indirect labeling of cDNAs was a modification of a method described elsewhere (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm). Briefly, cDNA probes were made from 2 µg of amplified RNA in a reaction volume of 30 µl containing 170 ng random hexamer primers, 0.2 mM 5-(3aminoallyl)-2_-deoxyuridine-5_-triphosphate (amino acid-dUTP; Sigma-Aldrich), 0.3 mM dTTP, 0.5 mM each dATP, dCTP, and dGTP, and 380 units of Superscript II reverse transcriptase (Life Technologies) incubated at 42oC for 120 minutes. After RNA hydrolysis, purified cDNA was combined with either Cy3 or Cy5 monoreactive fluors (Amersham Pharmacia) that covalently couple to the cDNA incorporated aminoallyl linker in the presence of 50 mM NaHCO3 (pH 9.0). The coupling reaction was quenched with Hydroxylamine and reference and experimental probes were combined, filtered, and competitively hybridized to microarrays under a coverslip for 16 h at 63°C. Slides were washed sequentially with 1X saline sodium citrate (SSC)/0.03% sodium dodecyl sulfate (SDS), 1X SSC, 0.2X SSC, 0.05X SSC, and spun dry. To account for dye bias, half of the biological experimental samples per strain were labeled with Cy3 dye and the reference with Cy5 dye, and in the other half the labeling was inversed. Hybridization procedures and parameters: The protocol and conditions used for hybridization, blocking and washing, including any post-processing steps such as staining. Reference and experimental probes were combined, filtered, and competitively hybridized to microarrays under a coverslip for 16 h at 63°C. Slides were washed sequentially with 1X saline sodium citrate (SSC)/0.03% sodium dodecyl sulfate (SDS), 1X SSC, 0.2X SSC, 0.05X SSC, and spun dry. Measurement data and specifications: Data o The raw data, i.e. scanner or imager and feature extraction output (providing the images is optional). The data should be related to the respective array designs (typically each row of the imager output should be related to a feature on the array – see Array Designs). Table 3. gpr file information Sample pool ID gpr file name GSM Number GenePix Results # GenePix Version Gal File Used 129-1 MuPr0294_129-1_ref_20040722.gpr GSM138319.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal 129-2 MuPr0286_ref_129-2_20040722.gpr GSM138315.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal 129-3 MuPr0293_ref_129-3_20040722.gpr GSM138318.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal 129-4 MuPr0290_129-4_ref_20040722.gpr GSM138317.gpr GenePix Results 3 Balb-1 MuPr0225_ref_Balb-1_20040607.gpr GSM138300.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal Balb-2 MuPr0240_Balb-2_ref_20040611.gpr GSM138306.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal Balb-3 MuPr0251_Balb-3_ref_20040611.gpr GSM138313.gpr GenePix Results 3 Balb-4 MuPr0250_ref_Balb-4_20040611.gpr GSM138312.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal C57-1 MuPr0228_ref_C57-1_20040607.gpr GSM138302.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal C57-2 MuPr0288_C57-2_ref_20040722.gpr GSM138316.gpr GenePix Results 3 C57-3 MuPr0241_C57-3_ref_20040611.gpr GSM138307.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal C57-4 MuPr0244_ref_C57-4_20040611.gpr GSM138309.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal DBA-1 MuPr0229_ref_DBA-1_20040607.gpr GSM138303.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal DBA-2 MuPr0236_DBA-2_ref_20040611.gpr GSM138305.gpr GenePix Results 3 DBA-3 MuPr0234_DBA-3_ref_20040611.gpr GSM138304.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal DBA-4 MuPr0249_ref_DBA-4_20040611.gpr GSM138311.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal FVB-1 MuPr0226_ref_FVB-1_20040607.gpr GSM138301.gpr GenePix Results 3 FVB-2 MuPr0254_FVB-2_ref_20040611.gpr GSM138314.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal FVB-3 MuPr0243_FVB-3_ref_20040611.gpr GSM138308.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal GSM138310.gpr GenePix Results 3 GenePix Pro 4.1 05-10-04 MPEDB_401-800.gal FVB-4 MuPr0246_ref_FVB-4_20040611.gpr o The normalized and summarized data, i.e., set of quantifications from several arrays upon which the authors base their conclusions (for gene expression experiments also known as gene expression data matrix and may consist of averaged normalized log ratios). The data should be related to the respective array designs (typically each row of the summarized data will be related to one biological annotation, such as clone name or specific oligo). See Normalized Log Ratios File. Data extraction and processing protocols, o Image scanning hardware and software, and processing procedures and parameters. : Fluorescent array images were collected for both Cy3 and Cy5 using a GenePix 4000B fluorescent scanner (Axon Instruments, Foster City, CA). The image intensity data were gridded and extracted using GenePix Pro 4.1 software. o Normalization, transformation and data selection procedures and parameters. For each array spot, the expression levels of the two fluorophores were obtained by subtracting median background intensity from median foreground intensity. A gene was only considered expressed if the fluorescence intensity of the corresponding spot was at least 6 foreground pixels greater than 4 standard deviations above background in at least half of the arrays per strain. Array spot not meeting these criteria were designated NA. For each gene, the logarithm base 2 ratios (referred henceforth as log ratios) of the two channels were calculated to quantify the relative expression levels of genes between the experimental and reference samples. To allow for inter-array comparisons, each array was normalized to remove systematic sources of variation. This was accomplished by means of a print-tip-specific intensity-based normalization method [35]. A scatter-plot smoother, which uses robust locally linear fits, was applied to capture the dependence of the log ratios on overall log-spot intensities. The log ratios were normalized by subtracting the fitted values based on the print-tip-specific scatter-plot smoother from the log ratios of experimental and control channels. After normalization, spots were removed from further analysis if they had more then 3 NA values or if they were in the lower third quantile of abundance across all 20 arrays.