Computational protocol: CD10-Equipped Melanoma Cells Acquire Highly Potent Tumorigenic Activity: A Plausible Explanation of Their Significance for a Poor Prognosis

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[…] Total RNA was isolated using an RNeasy Mini Kit (QIAGEN, Valencia, CA), and was purified by ethanol precipitation and dissolved in RNase-free water. This preparation was electrophoresed on an Experion System (Bio-Rad Laboratories), and the quality was confirmed by measuring the ratio of band intensity between 28 S and 18 S ribosomal RNA. The total RNA (250 ng) was converted to its biotinylated-cRNA in accordance with the manufacturer’s instructions (Illumina TotalPrep RNA Amplification Kit; Ambion, Carlsbad, CA, US). Briefly, the reverse transcription of extracted mRNA to first-strand cDNA was performed for 2 hours at 42°C, using an oligo (dT) primer bearing a T7 promoter and reverse transcriptase (ArrayScript; Ambion). To prepare second-strand cDNA, DNA polymerase I and ribonuclease H were added to the above reaction mixture, and the solution was further incubated for 2 hours at 16°C. After cDNA purification using a cDNA filter cartridge, the eluted cDNA was used as the template for in vitro transcription. This reaction was performed at 37°C for 14 hours in the presence of T7 RNA polymerase and NTP mix conjugated with biotin, yielding multiple copies of biotinylated antisense RNA to each mRNA in the sample. A total of 1.5 μg of biotinylated-cRNA was overlaid onto individual array spots of the human microarray chip (Illumina HumanHT-12 v4). The chip was hybridized at 58°C for 19 hours, washed, labeled with fluorescent reagent, and scanned using an array reader (BeadArray Reader; Illumina, San Diego, CA, US). The data on gene expression were compiled using Bead Studio software (Illumina).In the microarray analysis, average normalization was performed using Illumina software (Genome Studio v 1.8). If normalized expression values were below 0.1, then we replaced these values with 0.1. Probes with a detection P-value > 0.01 in all samples were removed. Statistical analyses were performed using R software (http://www.R-project.org). We calculated P-values and fold changes between CD10-A375 and mock-A375 groups. The significance of differences between the two groups was estimated using the package samr in R. The significantly differentially expressed genes were defined as those with P < 0.01 in a two-class unpaired Significance Analysis of Microarrays (SAM) t-test and fold change > 2 or < 0.5 between the two groups.A heat map was created using Mev4.6 for the 1,247 probes of genes significantly differentially expressed between CD10-A375 and mock-A375. The distance between the samples in the heat map was calculated using the Pearson correlation coefficient. Gene expression values were normalized by a Z-scaling method using a gene filter library with R. Gene Ontology annotation was assigned to significant genes identified by SAM using LSKB software (World Fusion Inc., Tokyo, Japan). The array data set was deposited in the Gene Expression Omnibus (series GSE62464).Fifteen representative genes identified by microarray were validated using qRT–PCR with commercially available primers, as shown in . Total RNA was reverse-transcribed with a first-strand cDNA synthesis kit for RT-PCR (PrimeScript RT Reagent Kit; Takara Bio Inc., Shiga, Japan), in accordance with the manufacturer’s instructions. For all samples, 50 ng of cDNA was used for qRT-PCR analyses. The reverse-transcribed cDNA was then subjected to qRT-PCR (SYBR Premix Ex Taq; Takara Bio Inc.) and thermal cycling (Mx3000P Real-time qPCR Systems; Stratagene, La Jolla, CA). The reaction conditions were denaturing at 95°C for 30 seconds, followed by 40 cycles of denaturing at 95°C for 5 seconds, and annealing and extending at 60°C for 20 seconds. The level of mRNA expression was estimated from the fluorescence intensity relative to β-actin (ACTB). […]

Pipeline specifications

Software tools Beadarray, SAM
Application Gene expression microarray analysis
Organisms Mus musculus, Homo sapiens
Diseases Melanoma, Neoplasms
Chemicals Etoposide, Thiorphan