BMC Genomics 2009, 10:150 http://www.biomedcentral.com/1471-2164/10/150 Isolation of CMs and videomicroscopy CMs were isolated from 12 week-old mouse hearts mounted for cardiac retrograde aortic perfusion as per- formed previously [11,38,39]. After fixing, the profiles of isolated CMs were visualized by autofluorescence (excita- tion 540 nm, emission 620 nm) and their image captured with a Retiga EXi monochrome camera (Qimaging, Sur- rey, BC, Canada). The area, length, width and width/ length ratio of ~100 CMs per heart were measured using the public domain Java image processing program ImageJ [40], as described previously [11]. Microarray experiments Total RNA was extracted, using the RNeasy minikit (Qiagen Canada, Mississauga, ON), from cardiac left ventricles obtained from 12 week-old male C57BL/6J or C57.YA that had undergone either castration or sham surgery between 3 and 4 weeks of age. All animals were killed between 9:00 and 10:00 AM. Four animals were used for each group, thus providing a total of 16 individual samples. Bioti- nylated probes were prepared from 50 ng of total RNA, using the Ambion Illumina TotalPrep RNA Amplification kit (Applied Biosystems, Streetsville, ON). The probes were hybridized to two Illumina MouseRef-8 v2.0 Bead- Chips that carry 8 microarrays each, containing 25,697 probes covering 19,728 non-redundant, well-annotated RefSeq sequences covered by either one single probe (79.3%), 2 probes (15%) or 3 or more probes (5.7%). Raw data (probe unnormalized intensity values) were obtained using the BeadStudio software (Illumina, San Diego, CA) and imported into the R 2.7.0 (UNIX) lan- guage/environment for normalization and analysis. Quantile normalization was performed using functions provided by the Affy package, as described [41]. Raw and normalized data have been deposited into the Gene Expression Omnibus (GEO) public depository (submis- sion GSE15354), in accordance with MIAME standards. To decrease the number of multiple comparisons across genes, undetected genes were eliminated from the dataset using Present/Absent calls, as recommended [42]. Detec- tion probability was estimated with the BeadStudio appli- cation by using the distribution of intensity values of negative probes. Transcripts were called as present when probability of detection was equal or higher than 0.96. Probes having a "present" call in at least 50% of samples belonging from any strain and/or surgery group were selected for further analyses. A linear model including genotype (strain) and treatment (castration) was fitted to normalized data: yij = μ + G + Tj + I(GxL)ij + eijk, where yij is i the normalized-transformed gene expression, μ is the population mean, Gi is the effect of ith genotype, Tj is the effect of jth castration treatment, I(GxT)ij is the effect of genotype by treatment interaction, and eijk is the residual effect. The significance of effects from strain, surgery and their interaction was tested by appropriate contrasts in a F- test between groups in a factorial ANOVA design. The F- tests were calculated with the James-Stein shrinkage esti- mate using information from neighboring probes [43]. P- values were calculated by performing 1000 permutation of samples to break their association to expression values, then corrected for multiple comparisons by adaptive false- discovery rate (FDR) transformation [44], using a 10% FDR cutoff. All computations were done with the R/ Maanova package v. 1.10.0 [45]. The set of probes with significant interaction effects, i.e. the "Interaction Genes set" was tested for enrichment of any KEGG pathway in the KEGG database http:// www.genome.ad.jp. Enrichment for KEGG pathways was tested by comparing the number of genes in a pathway to what would be expected by chance if the Interacting Genes set was a random sample from the list of genes present in samples. Using a hypergeometric function, this probabil- ity is calculated, which is equivalent to a Fisher's Exact Test. Genes with multiple probes in the microarrays were selected if at least one probe presented an FDR < 0.1 for the interaction term. Genes with multiple probes were counted only once in this analysis. Pathways with a p- value ≤ 0.05 were selected. Computations were performed with the GOstats package for R [46]. PCR and RT-PCR amplification of total RNA To verify which chrY genes were expressed in the heart and/or other tissues, total RNA was extracted from several tissues of adult C57BL/6J mice using the RNeasy minikit (Qiagen Canada, Mississauga, ON). The tissues were the left cardiac ventricles, quadriceps muscle, small and large intestine, brain, thymus, lungs, kidneys, pancreas, adrenal glands, testes, peritoneal fat and bladder. Complementary DNA was then synthesized (using 2 μg of total RNA from each tissue) using Superscript II reverse transcriptase (Invit- rogen Canada, Burlington, ON), and 100 ng of cDNA from each sample was then PCR amplified. Each primer pairs was designed to overlap at least one exon (with the exception of Sry, which is comprised of a single exon). Amplification products were visualized by UV illumina- tion of products electrophoresed in an agarose gel con- taining ethidium bromide. Primers used for amplification of chrY of cDNA reverse-transcribed from chrY gene mRNA transcripts are summarized in Additional file 3. Quantitative RT-PCR was performed to: 1) compare the abundance of chrY transcripts in hearts from C57BL/6J and C57.YA male mice; and 2) to validate results obtained by microarray experiments. For each primer pair, amplifi- cation was performed using reagents from the PCR Quant- iTect SYBR Green kit (Qiagen) and the Mx3005P thermocycler (Stratagene, La Jolla, CA). After 40 rounds of amplification, the specificity of each primer pair was veri- Page 9 of 11 (page number not for citation purposes)