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Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay.

ABSTRACT: BACKGROUND: Pseudorabies virus (PRV), a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. Real-time RT-PCR is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of RNA molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection. RESULTS: In this study, we have developed and applied a quantitative reverse transcriptase-based real-time PCR technique in order to profile transcription from the whole genome of PRV after lytic infection in porcine kidney cells. We calculated the relative expression ratios in a novel way, which allowed us to compare different PRV genes with respect to their expression dynamics, and to divide the PRV genes into distinct kinetic classes. This is the first publication on the whole-genome analysis of the gene expression of an alpha-herpesvirus by qRT2-PCR. We additionally established the kinetic properties of uncharacterized PRV genes and revised or confirmed data on PRV genes earlier examined by traditional methods such as Northern blot analysis. Our investigations revealed that genes with the same expression properties form clusters on the PRV genome: nested overlapping genes belong in the same kinetic class, while most convergent genes belong in different kinetic classes. Further, we detected inverse relationships as concerns the expressions of EP0 and IE180 mRNAs and their antisense partners. CONCLUSION: Most (if not all) PRV genes begin to be expressed from the onset of viral expression. No sharp boundary was found between the groups of early and late genes classified on the basis of their requirement for viral DNA synthesis. The expressions of the PRV genes were analyzed, categorized and compared by qRT2-PCR assay, with the average of the minimum cycle threshold used as a control for the calculation of a particular R value. In principle, this new calculation technique is applicable for the analysis of gene expression in all temporally changing genetic systems.

SUBMITTER: Tombacz D

PROVIDER: S-EPMC2775753 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay.

Tombácz Dóra D Tóth Judit S JS Petrovszki Pál P Boldogkoi Zsolt Z

BMC genomics 20091023

<h4>Background</h4>Pseudorabies virus (PRV), a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. Real-time RT-PCR is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of RNA molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection.<h4>Results</h4>In this study, we ...[more]

PMID: 19852823

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Project description:Predicting the clinical course of breast cancer is often difficult because it is a diverse disease comprised of many biological subtypes. Gene expression profiling by microarray analysis has identified breast cancer signatures that are important for prognosis and treatment. In the current article, we use microarray analysis and a real-time quantitative reverse-transcription (qRT)-PCR assay to risk-stratify breast cancers based on biological 'intrinsic' subtypes and proliferation.Gene sets were selected from microarray data to assess proliferation and to classify breast cancers into four different molecular subtypes, designated Luminal, Normal-like, HER2+/ER-, and Basal-like. One-hundred and twenty-three breast samples (117 invasive carcinomas, one fibroadenoma and five normal tissues) and three breast cancer cell lines were prospectively analyzed using a microarray (Agilent) and a qRT-PCR assay comprised of 53 genes. Biological subtypes were assigned from the microarray and qRT-PCR data by hierarchical clustering. A proliferation signature was used as a single meta-gene (log2 average of 14 genes) to predict outcome within the context of estrogen receptor status and biological 'intrinsic' subtype.We found that the qRT-PCR assay could determine the intrinsic subtype (93% concordance with microarray-based assignments) and that the intrinsic subtypes were predictive of outcome. The proliferation meta-gene provided additional prognostic information for patients with the Luminal subtype (P = 0.0012), and for patients with estrogen receptor-positive tumors (P = 3.4 x 10-6). High proliferation in the Luminal subtype conferred a 19-fold relative risk of relapse (confidence interval = 95%) compared with Luminal tumors with low proliferation.A real-time qRT-PCR assay can recapitulate microarray classifications of breast cancer and can risk-stratify patients using the intrinsic subtype and proliferation. The proliferation meta-gene offers an objective and quantitative measurement for grade and adds significant prognostic information to the biological subtypes.

Dataset Information

Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay.

Publications

Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay.

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