Design, validation and evaluation of a SYBR green-based quantitative PCR array for comprehensive analysis of adenovirus type 5 transcriptional patterns

Abstract

The adenoviral genome encodes coordinately expressed early and late gene transcriptional units that specify a complex collection of extensively spliced overlapping mRNAs. These complexities confound the generation of compatible, validated and optimized qPCR assays that permit comprehensive evaluation of adenoviral transcription. We have developed and evaluated a compilation of qPCR assays that represent the majority of the human adenovirus 5 (hAdV5) genome and allow for absolute and relative quantification of transcriptional activity. A panel of specific adenovirus gene primer pairs was designed through computational modeling to be compatible under a single reaction condition, precisely amplify spliced transcript products within each gene class, and not result in cellular or viral RNA/DNA background amplification. Primer pairs and reaction conditions were optimized to generate a single amplification product that was specific for its target amplicon with minimal intra-assay variability. The specificity of target amplicons was confirmed by dissociation curve analysis, gel electrophoresis and sequencing. In all, thirty-two primer sets representing specific gene products, as well as, pan early and late gene regions were validated under identical amplification conditions, thereby enabling a comprehensive assessment of adenoviral transcription within a single plate array. In order to generate positive control templates and to facilitate absolute quantification of gene expression, all target amplicons were cloned to create gene target-specific standards. These plasmid amplicon controls demonstrated that the SYBR qPCR assays exhibited optimal amplification efficiencies with a high sensitivity of detection to less than 10 copies and a linear amplification across at least eight orders of magnitude. The effectiveness and utility of the comprehensive adenoviral transcriptional array was assessed by investigating the changes in Ad5Wt gene expression at 72 versus 24 h post infection. Predictably, overall gene expression was globally increased at 72 h post infection; however, levels of E2 and Late transcripts exhibited the greatest increased expression, reflecting their necessity at this time point for genomic replication and virion assembly. Taken together, these data demonstrate that the adenoviral qPCR transcriptional array is a modular, scalable, and cost-effective method to comprehensively and accurately assess hAdV5 gene transcription. This array is broadly applicable to facilitate: adenoviral vector development; assessment of cell complementation of knockout viruses; antiviral mechanism of action evaluation; next-generation sequencing data validation.