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S. Di Cesare, A. Nantel, B. F. Fernandes, D. Abourbih, A. M. Saornil, M. N. Burnier, Jr.; Correlative Genetic Analysis of Cell Type Using DASL Gene Expression Profiling of Formalin-Fixed Paraffin Embedded Uveal Melanomas. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6184.
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© ARVO (1962-2015); The Authors (2016-present)
Fresh tissue from ocular melanomas are difficult to obtain for the purpose of performing microarray experiments on extracted nucleic acids. Present technology allows for extraction of total RNA from formalin-fixed paraffin embedded (FFPE) tissue to run genomic cancer arrays by the cDNA mediated Annealing Sectioning and Ligation (DASL) method. We aimed to correlate gene transcript differences between spindle and epithelioid uveal melanoma (UM) cell types in order to identify novel molecular targets for this particular malignancy.
A total of 44 FFPE UM were used. Each block was sectioned to a thickness of 10µm. The High Pure RNA Paraffin Kit (Roche) was used to extract total RNA from each of the 10µm sections. The quantity and quality of the total RNA extracted from each specimen was evaluated using a NanoDrop (ND-3300), qPCR, and an Agilent RNA 6000 Bioanalyzer. The expression of RPL13a, a ribosomal protein gene, for each sample was also assessed to evaluate the quality of RNA extracted from FFPE tissue (Illumina). All validated RNA samples were then processed and analyzed using the DASL method (Illumina). The cancer panel platform was used, which includes a pool of selected probe groups that targets 502 oncogenes. All samples were run in duplicate. Gene expression values generated from the array were analyzed using the Genespring GX software (Agilent).
A total average of 366 genes out of 502 were detected for all 44 UM samples analyzed (>0.99 using common detection score calculation). When the samples were normalized, we identified 25 genes with a statistically significant (p>0.05, Welch ANOVA Test) difference in transcript abundance between spindle and epithelioid cell types. The top five gene candidates (p> 0.001) include ERBB3>NEO1>ELK3>FLI1>IGF2R. In addition, each individual sample (spindle or epithelioid) exhibited distinct transcriptional profiles that can be separated on a principal components analysis (PCA) diagram.
To the best of our knowledge, this is the first time that usable RNA has ever been extracted from FFPE primary human UM. This is also the first time that archival tissue has been used to draw genetic transcriptional correlations with well established histo-pathological parameters. This data may lead to future customized therapeutic targets which may improve the survival rate of patients with ocular melanoma.
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