|Bono, James - Jim|
Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 10/3/2003
Publication Date: 11/9/2003
Citation: MILLER, L.C., CHITKO MCKOWN, C.G., BONO, J.L., LAEGREID, W.W., FOX, J.M. IDENTIFICATION OF RESPONSIVE GENES IN PRRSV-INFECTED PULMONARY MACROPHAGES USING SEMI-QUANTITATIVE REAL-TIME REVERSE TRANSCRIPTION PCR (RT-PCR). RESEARCH WORKERS IN ANIMAL DISEASES CONFERENCE PROCEEDINGS. 2003. ABSTRACT NO. 155.
Technical Abstract: Porcine reproductive and respiratory syndrome (PRRS) is caused by a virus which is classified as a member of the family Arteriviridae in the order Nidovirales. This syndrome causes highly significant losses to the swine industry worldwide. An important biological property of PRRSv is its primary replication in host alveolar macrophages (PAM), from where it may influence the host immune response. The aim of this study was to identify changes in PAM gene expression in response to infection with PRRSv. Preliminary results have demonstrated that there are very specific gene expression profiles to PRRSv infection of MARC-145 cells. Using microarray hybridization, it was found that with time there are outliers to linear RNA levels indicating changes in gene expression by more than two-fold between 10 and 24 hours (105 genes changed expression) post-infection. Over 20 genes had a differential of greater than four-fold. The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies. Relative expression levels were quantified by comparison of the relative amount of target sequence with a highly expressed reference gene. Using this approach, real-time PCR validated 17 of 21 (81%) genes in MARC-145 cells identified by DNA arrays. Real-time RT-PCR results suggest that genes identified by DNA arrays with a two- to four-fold difference in expression cannot be accepted as true or false without validation. Regardless of the gene expression profiling technology, once the sequence of the gene of interest is known, the real-time RT-PCR approach is well suited for validation of differential expression since it is quantitative and rapid and requires 1000-fold less RNA than other high throughput assays.