Two-Step Real-Time RT-PCR We next carried out studies to better evaluate the potential impact, directly or indirectly, of DNA methylation and/or histone deacetylation on the expression of specific genes that were selected based on microarray results. To do this, mouse cell lines (melan-a, 4C, 4C112 and 4C11+) were grown and treated as described above. Total cell RNA was extracted from 5AzaCdR and/or TSA-treated and untreated (control) cell monolayers using TRIzol reagent (Invitrogen). One microgram of RNA samples was reverse amplified for cDNA synthesis with integrated removal of genomic DNA contamination using QuantiTect Reverse Transcription Kit (QIAGEN), according to manufacturer’s instructions. The quality of the cDNA templates was checked by amplifying the endogenous reference gene Actb. With the aim of determining amplification efficiencies, we compared dilution series of reference and target genes from a reference cDNA template. Serial dilutions were amplified in two-step RT-qPCR and the Cq values obtained were used to construct standard curves for Actb, Fblim1, Hspb1, Serpine1 and Xist genes. The amplification efficiency of each gene was determined as follows: E = 10(-1/S) – 1, in which E corresponds to the efficiency and S to the slope of the standard curve. Next, equal amounts of each cDNA synthesized (200 ng/reaction) were quantified by real-time PCR in a Corbett Rotor-Gene 6000 Detection System version 1.7 using a SYBR green PCR master mix (QIAGEN) and a final concentration of specific forward and reverse primers at 400 nM. PCR cycling conditions were set to 5 minutes at 95uC followed by 40 cycles of 5 seconds at 95uC and 10 seconds at 60uC. A signal for non-template control (NTC) was evaluated as a control reaction since it enables detection of contamination. Transcripts presenting Cq values $ 35 were considered non-expressed. Because of the target(s) and the endogenous reference gene were amplified with comparable efficiencies (90%#E#110%), the relative quantification (RQ) of the amplicons was calculated according to 2-DDCq method. Mouse Actb was used as a single reference gene for normalizing cellular mRNA data because of its more stable expression among the previously untreated and treated cell lines than Gapdh gene (data not shown). To analyze the differences in the relative expression levels of genes among the sample groups representing distinct phases of melanocyte malignant transformation (melan-a, 4C, 4C112 and 4C11+), melan-a melanocyte lineage was taken as a reference sample and its average value was set arbitrarily to 1. For experiments involving treatment groups, untreated cell lines were taken as reference samples, and their average values were set arbitrarily to 1. Human Studies Human studies were performed with the aim of discussing the potential application of our mouse findings in a way more closely related to the pathogenesis of human melanomas. To do this, total mRNA was isolated from FM305 human primary melanocytes and metastatic melanoma cell lines (Mel-2, Mel-3, Mel-11, Mel-14 and Mel-33), and analyzed by two-step RT-qPCR, as described for mouse experiments. In this case, we selected a panel comprised by the genes CTCF, FBLIM1, HSPB1, NDRG2, NSD1, SERPINE1, SRC and VDR to address their potential use as biomarkers in identifying aggressive melanoma cells. To evaluate the differences in the relative expression levels of genes between the two sample groups (primary melanocytes and metastatic melanomas), the human primary melanocyte was used as a reference sample, and its average value was set arbitrarily to 1. For experiments involving treatment groups, untreated cell lines were taken as reference samples, and their average values were set arbitrarily to 1. Novel Candidate Biomarkers in Melanomas Unsupervised hierarchical clustering (Pearson correlation metric distance; average linkage algorithm) and relevance network prediction [59] of the genes validated in human primary melanocytes and five early-passage metastatic melanoma cell lines studied by RT-qPCR was performed using MeV software. Additionally, a conventional RT-PCR approach was used to assess the expression of XIST in two female human metastatic melanoma cell lines (Mel-11 and Mel-33) using a recombinant Platinum Taq DNA Polymerase (Invitrogen) and a final concen- tration of specific forward and reverse primers at 10 mM. PCR cycling conditions were set to 2 minutes at 94uC followed by 25 cycles of 30 seconds at 94uC, 30 seconds at 60uC, 30 seconds at 72uC and 7 minutes at 72uC. Primer Design for RT-qPCR To avoid amplification of genomic DNA, we designed mouse (Integrated DNA Technologies Inc., Coralville, IA) and human (Sigma-Aldrich) primer pairs flanking introns using IDT Primer- Quest and Primer3 web interface. All primer sequences are listed (Table S5). Statistical Analysis Statistical analysis of RT-qPCR results was performed by Student’s t test or One Way ANOVA test followed by the post-hoc Tukey. All statistics were carried out using GraphPad Prism software version 5 for Windows (GraphPad). Error bars represent SEM. The significance level was established at P,0.05. *P,0.05, **P,0.01, ***P,0.001. International Guidelines The Minimum Information About a Microarray Experiment (MIAME) as well as The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were used for the better experimental design and interpretation of microarray and qPCR results, respectively [60,61]. URLs ArrayExpress can be found at http://www.ebi.ac.uk/ arrayexpress/. IDT PrimerQuest can be found at http://www. idtdna.com/home/home.aspx. MeV can be found at http://www. tm4.org/mev/. PANTHER software can be found at http:// www.pantherdb.org/tools. Primer3 web interface can be found at http://frodo.wi.mit.edu/primer3/input.htm. RMAExpress can be found at http://rmaexpress.bmbolstad.com. Supporting Information Figure S1 Karyotype Evolutionary Description. Overall chromosomal rearrangements were shown in the representative images of the metaphases derived from karyotypes of (A) 4C112 non-metastatic, and (B) 4C11+ metastatic melanoma cell lines. Left panel: the raw image of a metaphase. Central panel: the classified image of the metaphase. Right panel: the inverted DAPI-banded image of the metaphase. Larger panel: the karyotype table of the metaphase. (TIF) Figure S2 Hierarchical Clustering of Samples and Dif- ferentially Expressed Genes Upon 5-aza-29-deoxycytidine Exposure Across the Melanoma Progression Spectrum. (TIF) Figure S3 Melanoma Stem Cell Markers Dynamically Regulated during melan-a Malignant Transformation. Green: Low expression; Red: High expression. (PPT) PLOS ONE | www.plosone.org 17 September 2012 | Volume 7 | Issue 9 | e44800