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Validation of Array‐based RNA Expression Profiles in Paraffin‐embedded Samples of Epstein‐Barr Virus‐related Malignancy

Epstein‐Barr virus (EBV) can be found in malignant cells and its viral genes represent targets for therapy and markers of tumor burden. In this study, the Nanostring system was used to evaluate 20 viral and 73 human RNAs in 13 paraffin embedded cell lines and in 16 carcinomas with matched non‐malignant tissue. RNA was successfully extracted from all specimens, and 100ng of RNA was used for analysis. Three different RNA purification strategies were compared. Crude lysates of RNA produced similar expression profiles but at lower levels than purified total nucleic acid. In addition, 3 spiked synthetic ERCC RNAs were successfully measured. Human tissue samples lacking EBV infection by the EBER in situ hybridization assay were essentially negative for all of the EBV transcripts. Unsupervised clustering revealed distinct and credible patterns of expression in epithelial vs lymphoid cell lines, in infected vs uninfected tissues or cell lines, and in tightly latent vs permissive forms of viral infection. This validation data suggests that RNA expression profiling of paraffin embedded tissue is feasible, and that use of purified nucleic acid promotes quality assurance. The synthetic ERCC reagents, when added during specimen processing, provide a resource for assessing quality of stored RNA.