The dietary isothiocyanate sulforaphane modulates gene expression and alternative gene splicing in a PTEN null preclinical murine model of prostate cancer

We obtained TRUS-guided prostate biopsy tissue from 21 volunteers who had previously received a diagnosis of HGPIN (mean time since diagnosis 135 ± 107 days, mean age 64 ± 4.3 years), and investigated PTEN and pAKT expression via immunohistochemichal staining in non-neoplastic prostate tissue. We found extensive heterogeneity in pAKT expression both among and within individuals (Figures 1 and 2). We also found extensive variation in expression of PTEN, with cells having both nuclear and cytoplasmic expression, solely cytoplasmic expression or neither nuclear nor cytoplasmic expression (Figures 1 and 3). While intense pAKT expression was associated with lack of both nuclear and cytoplasmic PTEN expression, a close inverse association between pAKT and PTEN expression was not apparent. In particular, epithelial cells from atrophic glands often had both pAKT and PTEN expression (Figure 1).

As an initial investigation into the potential interaction between PTEN deletion and SF, we quantified the effect of SF on the growth and proliferation of a human cancerous prostate cell line, PC3, which is deficient in PTEN and has high constitutive expression of pAKT [15], compared to the non-cancerous cell line PNT1A, which expresses PTEN and a low constitutive expression of pAKT. We found that at low concentrations, SF enhanced the growth and proliferation of PNTIA cells, while inhibited the growth and proliferation of PC3 cells. Both cell lines were inhibited at higher supraphysiological concentrations of SF (Figure 4).

To explore the interaction between PTEN expression and activity of SF specifically, we used PTEN^L/L;PB-Cre4 mice. In this model, segregation of Cre within a backcross generation results in PTEN deficient ('null') and wild type (WT) PTEN mice within the same litter, enabling the specific effects of PTEN deletion to be investigated [14]. We supplemented the diet of PTEN null mice and their WT littermates with either 0.1 μMol SF/g diet (low SF diet, LSF) or 1 μMol SF/g diet (high SF diet, HSF) and compared it to mice fed a control diet without added SF.

WT mice had normal prostate histology. However, at eight weeks foci of mild epithelial hyperplasia were observed in some sections, although they were not significantly associated with diet. PTEN deletion at both five and eight weeks was confirmed through RT-PCR analyses of prostate tissue (data not shown) and immunohistochemichal staining (Figure 5). Histopathological development of murine prostate intraepithelial neoplasia (mPIN) due to PTEN deletion was very similar to that previously described [14]. PTEN null prostate tissue from five-week old mice exhibited epithelial hyperplasia and some foci with early indication of mPIN which also showed increased pAKT expression. This association was more prominent at eight-weeks, where pAKT was highly expressed in all epithelial cells, with most glands exhibiting mPIN. Ki67 was widely expressed at both five and eight weeks in PTEN null prostate tissue but expression of phosphorylated mTOR was only detected at eight weeks (Figure 6). Diet did not have an obvious effect on the appearance of mPIN or expression of these proteins.

PTEN deletion resulted in multiple changes in gene expression at both five weeks and eight weeks (Table 1), associated with epithelial hyperplasia and mPIN, respectively. At five weeks, the PTEN deletion resulted in changes in expression of 198 transcripts (adjusted p < 0.05), while at eight weeks the deletion resulted in changes in expression of 989 transcripts (Table 1, Additional file 1 - S1). These are designated '5-week and 8-week PTEN signature genes'. 135 transcripts were common between these two lists of genes, which was significantly greater than the 11 transcripts that would have been expected by chance (Fishers exact test, p = 5.8 × 10^-44). A previous study reported changes in gene expression in the same mouse model between wild type prostate tissue and invasive adenocarcinoma in PTEN null prostate tissue obtained 26-29 weeks after birth, in which there were changes in expression of 1041 out of a possible 10290 genes/ESTs that were present on a custom-made cDNA array [14]. We compared this list with our 5-week and 8-week PTEN signature gene lists for which we had Unigene designations (193 and 963 respectively), but found little overlap between either the 5 week PTEN signature list or the 8 week PTEN signature list with the gene list obtained from analyses of tissue after 26-29 weeks (Figure 7). The five genes that were in common in all three lists are chemokine (C-C motif) ligand 20 (Ccl20), extracellular proteinase inhibitor (Expi), G protein-coupled receptor family C group 5 member A (Gprc5a), CD24a antigen (Cd24a) and calcium and integrin binding 1 (calmyrin, Cib1). It is therefore likely that PTEN deletion initiates a transcriptional cascade, in which genes that are altered in their expression in the early phase are distinct from that in the later phases associated with metastatic disease.

We found very few changes in prostate transcript expression induced by SF in the WT mice at 5 or 8 weeks of age (Table 1). In contrast, there were many dose-dependent changes in gene expression induced by SF in the PTEN null background (Table 1), suggesting that the effects of SF are dependent upon activation of PI3K/AKT signalling pathway due to PTEN deletion. This would explain how SF may have selective effects towards cells that are liable to proliferate. For further insight into the changes induced by the HSF diet, we undertook functional analyses with GenMAPP and found that there was a high frequency of genes specifically associated with PI3K/AKT mediated metabolic processes, such as amino acid, fatty acid, glucose metabolism and apoptosis (Table 2). When we compared the changes in expression of the genes induced by HSF to those that occurred in the PTEN null background compared to the WT background, we found that there was an inverse association. This indicates that the SF was able to suppress pAKT-mediated gene expression (Figure 8).

One rationale of feeding SF to PTEN knock out mice is that it is a model for the potential protective effects of broccoli on the development of prostate cancer in humans, partially validated by our observations that PTEN deletion may occur early in human prostate carcinogenesis (Figure 1). We thus sought to see if there was a significant overlap in the genes induced by SF in the PTEN null mice with previously obtained data on the changes in gene expression in sequential prostate biopsies of men who were deemed to be at risk of prostate cancer following a 12 month dietary intervention with either broccoli or peas, as previously described [13]. In this study, men who had previously received a diagnosis of HGPIN, and exhibited heterogeneity in expression of PTEN and pAKT (Figures 1 and 2), were randomized into two dietary intervention groups, in which they were required to consume either 400 g of peas per week or 400 g of high glucosinolate broccoli per week for 12 months but to otherwise follow their normal diet which was quantified through the use of diet diaries. Global gene expression was quantified through the use of human Affymetrix arrays in sequential biopsies obtained before and after the intervention from each of the volunteers, as previously described. We converted mouse genes that changed in expression following SF intervention into their human orthologues using the HomoloGene database at the NCBI website. We initially undertook comparisons between the human 156 Affymetrix probe sets orthologues of the 80 mouse genes that were altered in expression by HSF after five weeks (Table 1) with human genes that were altered in expression in prostate biopsy samples taken from volunteers before and after a 12-month intervention with the broccoli-enriched diet (5859 Affymetrix probesets). We found that 30 probesets were common to both lists, which was significantly greater (p < 0.002) than the 16 probesets that we would have expected by chance. When we compared the changes in gene expression in a similar group of men whose diet had been enriched with peas (4947 Affymetrix probesets), we found that 22 probesets were common to both lists, which was also significantly greater than that which we would expect by chance (14 probesets, p = 0.023). When we combined the three data sets (Figure 9), we found that 19 probesets were common to the three data sets. When we repeated the analysis with the 72 mouse genes that were altered in expression after eight weeks, we found no significant overlaps with the human data.

We then compared gene expression profiles between WT mice fed the control diet and PTEN null mice that had been fed either the control diet or diets supplemented with SF to investigate the combined effects of PTEN deletion and diet. When tissue was analysed at five weeks of age, it was apparent that the addition of SF to the diet had two effects. The LSF diet reduced the number of PTEN signature gene changes from 198 to 66, while inducing changes in expression of 221 additional genes that had not previously been perturbed by the PTEN deletion. These additional gene changes induced by LSF were associated with phosphate transport, cell adhesion and extracellular matrix organisation (Additional file 2 - Table S2). The HSF diet suppressed all changes in gene expression that had previously been induced by the PTEN deletion, and did not induce any additional changes in gene expression (Table 1).

The lack of statistically significant differences in gene expression between WT mice and PTEN null mice fed the HSF diet appeared inconsistent with epithelial hyperplasia observed in these mice at five weeks of age. We therefore compared the extent of the magnitude of changes in expression of the PTEN signature genes induced by the PTEN deletion with the control diet to that when the diet was supplemented with SF. We found that the HSF diet had not entirely suppressed changes in the PTEN signature genes, but attenuated the extent of the changes by about 33% (Figure 10A, Additional file 1 - Table S1), so that none of them alone reach statistical significance. Likewise, the LSF suppressed the extent of changes in the PTEN signature genes by 23% (Additional file 1 - Table S1). As there was similar suppression in all the PTEN signature genes, regardless of whether they had been up- or down-regulated or their ontological association, it is likely that SF has a direct effect on suppressing PI3K/AKT signalling. However, in this model the extent of suppression of PTEN null-mediated gene expression was insufficient to suppress pathological changes in prostate tissue.

When gene expression profiles were analysed in prostate tissue from eight week old mice, SF could again be observed to have two effects. First, the LSF and HSF diets reduced the number of PTEN signature gene changes from 989 to 723 and 761, respectively (Figure 11). This more modest reduction was also reflected in the similarity of changes in the magnitude of expression of the majority of the 989 genes in PTEN null mice fed either the control diet, or diets supplemented with LSF or HSF (Figure 10B). However, both SF diets attenuated the expression of a subset of the PTEN signature genes involved in positive regulation of cell differentiation, cell division and post-translational protein modification (Additional file 3 - Table S3). Secondly, the addition of SF to the diet of PTEN null mice resulted in changes in the expression of additional genes (Figure 11, Additional file 4 - Table S4). Pathway analyses of the genes that were altered in expression due to the PTEN deletion and its interaction with SF is summarised in Table 3. As would be expected, certain pathways, associated with changes induced by the PTEN deletion were common to PTEN null prostate tissue regardless of diet, such as immune and inflammatory response, whereas mice that had diets supplemented with SF had additional changes in pathways associated with cell cycle regulation and apoptosis (Table 3). The change in expression of genes within these pathways were indicative of SF inducing cell cycle arrest and apoptosis (Additional file 5 - Table S5; Additional file 6 - Table S6). For example, the up regulation of cyclin B1 and the down regulation of cyclin D2 are consistent with cell cycle arrest, and the two fold increase in expression of caspases 3 and 7 are consistent with the induction of caspase-induced apoptosis. It is notable that we did not observe significant up regulation of Nrf2 regulated genes.

As an overview, the data on changes in gene expression suggest that SF has two effects: it can both suppress PTEN deletion - mediated gene transcription, such as that observed at five weeks, particularly by the HSF diet, as well as inducing additional changes in gene expression. The balance between these two effects, and thus the overall changes in gene expression, depends upon the age of the mouse and the concentration of SF.

The development of high density exon arrays facilitates the global analyses of alternative splicing events. Several statistical approaches have been developed to analyse the large and complex data sets arising from the use of these arrays [16], of which we used FIRMA ('Finding Isoforms using Robust Multichip Analysis') [17], specifically developed for analysing Affymetrix exon array data and implemented in the aroma.affymetrix package of R/Bioconductor [18]. Briefly, FIRMA scores each exon as to whether its probes deviate from the expected transcript expression level and are used to perform further statistics to identify alternative splicing events.

Initially, we compared changes in exon FIRMA profiles induced by SF in WT or PTEN null genetic backgrounds. In a similar manner to that observed for transcriptional changes, SF induced only potential splicing events in the PTEN null background implying the need for activated PI3K/AKT signalling (Table 4). However, the greatest number of exon skipping/inclusion was evident when comparisons were made between WT mice and PTEN null mice whose diets had been supplemented with SF (Table 4, Additional file 7 - Table S7), indicating that SF interacts with the PTEN deletion to induce alternative splicing. The functional pathways of the genes that were potentially alternatively spliced by SF are shown in Additional file 8 (Table S8). A large proportion of genes for which there was indication of alternative splicing also showed perturbed overall transcript expression by SF (74% for HSF and 47% for LSF), suggesting that alternative splicing by SF is coupled to transcription.

We selected DMBT1 (Deleted in Malignant Brain Tumors 1), a gene involved in innate immunity, cell differentiation and tumour suppression [19], to explore further the effects of PTEN genotype and SF on gene expression and splicing. FIRMA analyses suggested significant effects of PTEN deletion and SF on splicing of several exons (Figure 12) that was consistent with two known splice variants of DMBT1, the shorter isoform, designated as DMBT1-201 which is missing exons 1, 2 and 7 and 10, and the longer isoform, DMBT1-202 which contains these exons (Figure 13A). We designed PCR primers within exons 1, 3, 4 and 10 which would distinguish the two isofoms (Figure 13A).

At five weeks of age, neither PTEN nor SF had an effect on overall expression of the DMBT1 transcript (Figure 13B). However, we found that all WT mice, regardless of diet, lacked amplification of PCR products due to the likely absence of exons 1 and 10, consistent with the expression of the shorter isoform. In contrast, all PTEN null mice fed the control diet had expression of the longer isoform (Figure 13C(i) and (iii)). However, five-week old PTEN null mice fed the SF diets varied in which isoform they expressed (Figure 13C(i) and (iii)), indicative of a possible effect of SF on suppression of alternative splicing induced by PTEN deletion, similar to the manner by which SF can suppress PTEN deletion-mediated gene expression at five weeks (Figures 8 and 10A, Additional file 1 - Table S1).

After eight weeks, there was a significant effect on expression of the DMBT1 transcript by PTEN genotype (p > 0.001), SF (p = 0.008), and by a PTENxSF interaction (p = 0.007, Figure 13B), illustrating the manner by which PTEN and diet can interact to determine gene expression, evident from Table 1. Eight-week old WT mice, regardless of diet, express the short isoform whereas PTEN null mice, again regardless of diet, all expressed the long isoform (Figure 13C(ii) and (iv)). It is not possible to say from these preliminary analyses whether at eight weeks the enhanced transcription of DMBT1 by SF induces the PTEN-mediated alternative splicing. However, the large number of genes within PTEN null mice on SF diets that express alternatively spliced transcripts compared to WT mice on the control diet (Table 4, Additional file 7 - Table S7) suggests that this may be the case. Further studies of the interaction between PTEN genotype and diet are required.

This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the East Norfolk & Waveney Research Governance Committee and the Norfolk Research Ethics Committee (references 05/Q0101/9 and 09/H0311/96). All patients provided written informed consent for the collection of samples and subsequent analyses.

All animals were handled in strict accordance with good animal practice and all animal work was approved by the University of East Anglia Animal Ethics Committees and covered by the appropriate licences under the UK Home Office Animal Procedures Act, 1986 (PPL 80/1799).

Trans-Rectal Ultra Sound (TRUS)-guided needle biopsy tissues were obtained from men who had previously received a diagnosis of high grade prostatic intraepithelial neoplasia (HGPIN) via the Urology Clinic at the Norfolk and Norwich University Hospital as previously described [13]. Briefly, RNA was extracted from whole biopsy tissues for gene expression analysis and adjacent biopsies were evaluated by histology. Although the latter did not contain neoplastic cells, we cannot exclude the presence of these cells in the biopsies used for gene expression analysis. Details of the volunteers (age, BMI, PSA, GSTM1 genotype) are as previously described [13].

The human post-pubertal prostate normal (PNT1A) cell line and the human Caucasian prostate adenocarcinoma (PC3) cell line were obtained from the European Collection of Cell Cultures (ECACC). PNT1A and PC3 cells were routinely cultured as monolayers in RPMI-1640 and HAMS media, respectively, supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were grown to 70-80% confluence before incubation in complete media with various concentrations of SF. Cell viability was determined following a 24 h treatment with either SF or vehicle DMSO using the WST-1 Cell Proliferation Reagent (Roche Applied Science). Cell proliferation was assessed by ELISA following BrdU incorporation (Roche Applied Science).

PTEN^L/L;C⁺ mice were generated by crossing ARR2Probasin-Cre transgenic mice, PB-Cre4 [56], to PTEN^L/L mice [57] as described before [14]. Only F2 generation male offspring (PTEN^L/L;C⁺ ) and their littermate controls (PTEN^L/L;C^-) were used in this study. For simplicity PTEN^L/L;C⁺ mice will be referred to as PTEN null and PTEN^L/L;C^- mice as wild type (WT). PB-Cre4 mice (strain B6.D2-Tg(Pbsn-cre)4Prb) were obtained from the Mouse Models of Human Cancers Consortium (MMHCC) of the National Cancer Institute, US, and PTEN^L/L mice (strain C;129S4-PTEN ^tm1Hwu /J) from the Jackson Laboratories (http://jaxmice.jax.org). Mice were housed in a room with controlled humidity, temperature and light within the Disease Modelling Unit at the University of East Anglia.

Genotyping for the Cre recombinase and PTEN genes was performed by PCR using tail tip DNA. DNA was extracted using the QIAGEN DNA blood and tissue kit (QIAGEN). For the PB-Cre4 genotyping we used two sets of primers, one specific to the transgene producing a 199 bp product (5'-ACC AGC CAG CTA TCA ACT CG-3' and 5'-TTA CAT TGG TCC AGC CAC C-3') and the other as an internal control producing a 324 bp product (5'-CTA GGC CAC AGA ATT GAA AGA TCT-3' and 5'-GTA GGT GGA AAT TCT AGC ATC ATC C-3'). Excision of exon 5 of the PTEN gene was determined by the size of the product from the PCR amplification using the primers 5'-ACT CAA GGC AGG GAT GAG C-3' and 5'- GCC CCG ATG CAA TAA ATA TG-3', where 1.2 Kb results from a wild-type PTEN allele and 1.3 kb from a mutant allele.

Animal feed consisted of basal diet (AIN-93G, control diet) or basal diet enriched with either 0.1 μMol SF/g diet (LSF diet) or 1 μMol SF/g diet (HSF diet). D, L-SF was purchased from LKT laboratories (St. Paul, MN, USA) and was incorporated into the basal diet by Harlan Teklad (Madison, WI, USA). SF levels were confirmed in the diets by LC-MS as described previously [58]. It is noteworthy that that we chose lower SF levels compared to previous studies, ranging between 1.2-3.4 μMol SF/g [4, 21], so that the intake would reflect more physiological dietary exposure. Homozygous male PTEN null mice and their littermate controls were randomly assigned to the experimental and control diets, which they received from weaning ad libitum for a duration of either two weeks or five weeks. Animals were sacrificed by CO₂ asphyxiation and cervical dislocation at five and eight weeks of age. At the time of sacrifice, the total mouse weight was recorded. Mouse prostates were removed immediately and either placed in RNAlater^® and stored at -20°C for subsequent expression analysis (n = 3 per group) or fixed in 10% formalin, embedded in paraffin wax, and used for histopathological evaluations and immunohistochemichal (IHC) staining (n = 3-5 per group).

Dissected tissue samples from mouse and biopsy samples from human volunteers were fixed in 10% formal-saline, embedded in paraffin wax and sections approximately 4 μm thick were cut. One slide from each tissue sample was stained with hematoxylin and eosin (H&E). For immunohistochemical assessment of both the mouse and the human tissue samples, the slides were deparaffinised in three changes of xylene and rehydrated through graded ethanols (100% to 50%). Heat-induced antigen retrieval was performed using citrate buffer pH 6.0 (Dako UK Ltd., Ely, Cambridgeshire, UK). Endogenous peroxidises were quenched using 3% hydrogen peroxide in PBS or TBS and then blocked with 1%BSA in PBST or 5% normal goat serum in TBST appropriate to manufacturer's instructions for each primary antibody. Primary antibodies were diluted in appropriate buffer according to the supplier's instructions: Anti-mouse Ki67 rat monoclonal (Dako, #M7249) diluted 1:50 in PBST; Anti-PTEN(D4.3) rabbit monoclonal diluted 1:125 in SignalStain^® Antibody Diluent(Cell Signaling, New England Biolabs, Hitchin, UK); Anti-pAKT (Ser473) (D9E) rabbit monoclonal diluted 1:50 in SignalStain^®; Anti-Phospho-mTOR (Ser2448)(49F9) rabbit monoclonal diluted 1:50 in TBST; (Cell Signaling antibodies: #9188; #4060 and #2976 respectively). All were incubated at 37°C for 30 mins. Detection was performed using the appropriate secondary antibody with Vector ABC Elite kits and DAB substrate kit (Vector Laboratories, Peterborough, UK).

All slides were counterstained with Mayer's haematoxylin (Surgipath Europe Ltd, Peterborough, UK), dehydrated through graded ethanols (50% to 100%), and mounted with cover slips. Slides were visualised using an Olympus BX60 (Olympus, Japan) microscope with ProgRes^® Capture Pro 2.1 software (Jenoptik, Germany). Histopathological assessment was performed according to the classification of the Bar Harbor Meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee [59]. Stained sections were evaluated by two independent researchers. The immunohistochemical quickscore (Q) was determined for each by multiplying the estimated percentage of positively stained cells (P) by the intensity of staining (I). Estimates of intensity were scored as follows: 1 for weak staining; 2 for moderate staining and 3 for strong staining (Q = P × I; maximum 300).

RNA from mouse prostate tissue stored in RNAlater^® was isolated using RNeasy Mini kit (QIAGEN) and gene expression profiling was performed using the Affymetrix GeneChip^® Mouse Exon 1.0 ST Array (Affymetrix, Santa Clara, CA) at the Nottingham Arabidopsis Stock Centre (Nottingham, UK) according to the Affymetrix protocols. This array is a whole-genome array, containing 1.2 million probesets of four Perfect Match (PM) probes each. A total of 36 arrays were hybridised (3 mice X 2 genotypes (WT and PTENnull) X 3 diets X 2 time-points). Data were analysed using R/Bioconductor [60] and the aroma.affymetrix package [18]. Linear probe level models were fit to RMA-background corrected and quantile normalised data to get gene- or exon-level summaries. For annotation we used the current custom CDF file available at the aroma.affymetrix website containing the core probesets (17,831 transcript clusters; 224,053 probesets). One array was identified as an outlier and was removed from further analysis. Subsequent statistical data analysis to identify differentially expressed genes was performed using limma [61], and evaluation of alternative splicing using FIRMA [17] packages. Genes were identified as differentially expressed at different Benjamini and Hochberg adjusted p-values. Alternative splicing events were identified if the difference between FIRMA scores of two groups was statistically significant after Benjamini and Hochberg adjustment. For the plotting of probeset-level data for FIRMA analyses of exon array data we used the Bioconductor GenomeGraphs package [62]. To identify pathways that were the most over-presented in the lists of differentially expressed or alternative spliced genes, functional analyses using MAPPFinder and GenMAPP v2.1 were performed http://​www.​genmapp.​org/​[63]. The Database for Annotation, Visualization and Integrated Discovery v6 (DAVID; http://​david.​abcc.​ncifcrf.​gov/​) [64] was used to identify Gene Ontology (GO) categories associated with specific gene lists (Additional file 2 - Table S2; Additional file 3 - Table S3). Microarray data generated in this study are compliant to MIAME criteria and are publicly available through ArrayExpress (Accession E-MEXP-2469).

Total RNA from all the mice was reverse transcribed into cDNA using random primer synthesis from the High-Capacity cDNA Reverse Transcription kit according to the manufacturer's instructions (Applied Biosystems, UK). Primer pairs for the locus flanking exon 2 were 5'- TTGTGGGGTCAAATTCTGTCT-3' and 5'-CTCCAGCATCTTCCTGGTGT-3' and for the locus flanking exon 7 were 5'-CTCAAACAAGCAGTCCCACA-3' and 5'-GTCCCTCCTGGATTCCACC-3'. PCR reactions were carried out in a total volume of 50 μl consisting of 1× green GoTaq^® Flexi PCR Buffer, 0.2 mM dNTPs, 0.2 μM primer, 1 mM MgCl₂, 0.025 units of GoTaq^® Flexi DNA Polymerase (Promega, UK). Cycling conditions consisted of 2 min at 95°C for initial denaturation, 30 cycles of denaturation for 30 sec at 95°C, annealing for 1 min at 60°C for exon 2 and 59°C for exon 7, and extension for 1 min at 72°C, followed by 5 min final extension at 72°C. Amplified products were separated in 2% (w/v) agarose gels using 1 μg of the 50 bp DNA ladder (Invitrogen, UK) as a size marker and visualised under UV following ethidium bromide staining.

To estimate whether the overlap between human orthologues of mouse genes induced by SF and human genes induced by a dietary intervention was significant, we initially assumed that the total number of probes from the human Affymetrix U133 Plus 2.0 array and equivalent probes from the mouse Affymetrix Exon 1.0 ST array would be similar. We then performed a Monte Carlo simulation to assess the number of expected possible probes that both change significantly in the mouse (measured probability = 0.000467) and the human (measured probability = 0.10716). This expected number was compared to the measured number using a Binomial test in R [65].