title: Analysis of Viral Promoter Usage in EBV-Infected Cell Lines: A Comparison of qPCR Following Conventional RNA Isolation and Nuclear Run-On Assay.
abstract: To interpret the results of an epigenetic analysis in gene expression studies, it is essential to characterize the activity of the relevant promoters. According to the literature, real-time PCR assay is the most widely used method for the determination of latent EBV promoter usage. Here we describe two alternative approaches to measure the activity of viral promoters in cell lines carrying latent EBV episomes. The widespread typical approach relies on total cellular RNA isolation, whereas the nuclear run-on assay described here is based on the initial isolation of nuclei, followed by in vitro transcription in the presence of biotinylated-UTP, and purification of RNA transcripts using avidin-coated magnetic beads. Finally, both methods apply reverse transcription-based real-time PCR (i.e., quantitative polymerase chain reaction, qPCR) to quantitatively measure the amount of specific transcripts. We shall describe these methods step by step and demonstrate their use for the determination of EBER1 promoter activity in EBV-positive cell lines.
authors.affiliation: RT-Europe Nonprofit Research Ltd., Vár 2., Dunacsunyi ut, Szigetköz Building, Mosonmagyaróvár, H-9200, Hungary.
authors.forename: Kalman
authors.initials: K
authors.lastname: Szenthe
authors.affiliation: RT-Europe Nonprofit Research Ltd., Vár 2., Dunacsunyi ut, Szigetköz Building, Mosonmagyaróvár, H-9200, Hungary. fbanati@yahoo.co.uk.
authors.forename: Ferenc
authors.initials: F
authors.lastname: Banati
mesh_terms: D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D014158:Transcription, Genetic; D017735:Virus Latency / Q000235:genetics
chemical_list: C099537:Epstein-Barr virus encoded RNA 1; D012367:RNA, Viral
keywords: Gene expression; Latent EBV promoter; Nuclear run-on assay; Quantitative PCR; Reverse transcription