The only two miRNAs that were down-regulated in TH-MYCN tumors from TH-MYCN/p53ERTAM mice were miR-125b-3p and miR-676. No specific role has thus far been ascribed to miR-676 in human neuroblastoma, which has a seed region that is not identical between the two species. Interestingly, miR-125b has been identified as both a target of p53 [47] and a regulator of the p53 transcript itself [48]. miR-125b inhibits neuroblastoma cell proliferation and promotes cell differentiation in in vitro models [49,50], so that down-regulation of this miRNA could potentially be contributing towards the more aggressive phenotype of the TH- MYCN/p53-ERTAM tumor phenotype. This is an interesting possibility requiring further studies. Although many of the differentially expressed miRNAs were previously identified as being associated with human neuroblas- toma (Table 1), this study identifies additional miRNAs not previously correlated with this pathology, indicating a potential role in neuroblastoma pathogenesis. Indeed, many of the most significantly differentiated miRNAs in mouse tumors versus adrenal tissue have never been associated with any form of cancer to date (Table S2). Functional studies are required to corroborate any role that these miRNAs might have in tumorigenesis. Lastly we demonstrate that, despite some differ- ences among the two species, the mouse model recapitulates a pattern of miRNA expression that is dependent on MYCN expression and which is similar to the one observed in human MNA tumors. Our results further support the involvement of miRNAs in neuroblastoma, validating the utility of TH-MYCN mouse model for studying the role of miRNAs in this pathology and for possible pre-clinical trials based on miRNA agonist/ antagonist molecules. Materials and Methods Transgenic mice and samples collection TH-MYCN mice were maintained in hemizygotic matings [26]. TH-MYCN/p53ERTAM animals were obtained serially crossing TH-MYCN+/2 and p53-ER +/2 mice [25]. Adrenals were obtained from mice 7 day post birth, while tumors were collected after the onset of the tumor pathology (.40 days post birth). All tissue samples were either frozen in liquid nitrogen or submerged in RNAlater RNA Stabilization Reagent (Qiagen, Crawley, UK) and stored at 280uC. All animals were handled in accordance with institutional guidelines for safe and ethical treatment of mice. The protocol was approved by the U.K. Home Office (License number PPL70/6882). RNA isolation Total RNA extraction was performed using RNeasy (Qiagen), miRNeasy (Qiagen), or mirVanaTM miRNA Isolation Kit (Ambion, Austin, Texas) following the manufacturer guidelines. Reverse transcription For messanger RNA (mRNA) cDNA synthesis, total RNA was reverse transcribed using random primers and a TaqMan Reverse Transcription kit (Applied Byosistems, Foster City, CA). Up to 762 rodent mature miRNAs were converted to cDNA (381 miRNAs processed simultaneously per reaction) using the MegaplexTM RT Rodent Pool (Applied Biosystems). Quantitative PCR for gene expression The level of endogenous p53 was analyzed using primers specifically designed for targeting exon 10 and 11 of the transcript (sequences of primers available on request) and SYBRH Green PCR Master Mix (Applied Biosystem). mRNA quantifications miRNA Expression Profiling of Murine Neuroblastoma were normalized to the housekeeping gene b-actin. Relative quantification of genes expression was determined using the comparative cycle threshold method (22DDCT). Pre-amplification and quantitative PCR of miRNAs Megaplex retro transcription product was pre-amplified using TaqMan PreAmp Master Mix and MegaplexTM PreAmp Primers, Rodent Pool (Applied Biosystems). The miRNA profile of each cDNA sample was obtained using 384-well microfluidic cards (TaqManH MicroRNA Array v2.0, Applied Biosystems). As instrument and liquid handling variations were shown to be minimal, no PCR replicates were measured. All quantitative PCR (qPCR) were carried out on the 7900 HT Fast Real-time System (Applied Biosystems). Data analysis Since the presence of a single molecule of target lead to a Ct value of 35, all miRNAs with Ct values greater than 35 were considered not expressed. The mean Ct of a sample was subtracted from the Ct value of each miRNA among that sample (mean centered normalization) before calculating relative expres- sion values [51]. Normalized relative expression (NRQ) of miRNA was calculated with reference to the Ctmax (maximum Ct value for an individual miRNA across all samples) using: NRQ = 2(Ctmax2Ct). Significance testing The statistical significance of miRNA differential expression over sample classes was evaluated by assigning p-values based on the non-parametric Wilcoxon Rank Sum Test. P-values were corrected for multiple comparisons using the Bonferroni method. The statistical significance of the enrichment for a specific genotype among the groups generated by the hierarchical clustering was evaluated using the chi-squared test (x2-test) on the frequency of observed samples compared to the expected random distribution. The statistical significance of the overlap between the lists of miRNAs was evaluated using the hypergeo- metric distribution [33]. miRNA homology identification An in-house developed Java (v6.0) software was used for comparing the sequences (retrieved from miRBase v16) of human and mouse mature miRNAs. We considered as homologue only miRNAs whose mature transcript showed perfect identity in both length and nucleotide sequence among the two species. Cluster analysis and visualization Hierarchical clustering, heatmap generation, principal compo- nent analysis (PCA) and the loadings of PCA were performed using ‘‘hclust’’ (agglomeration method complete based on Spearman rank correlation coefficient), ‘‘heatmap.2’’, ‘‘prcomp’’ and ‘‘pcs$rotation’’ functions of the R statistical computing language v2.13.0. Supporting Information Figure S1 Comparison of hematoxylin and eosin stained adrenal glands from wild-type and transgenic mice at day 7 after birth. The gross size is similar in transgenic versus wild-type mice and there is no evidence of changes in cell morphology indicative of hyperplasia or the development of early proliferative intra-adrenal preneoplastic foci. (TIF) PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28356