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Chronic Fatigue Syndrome > Publications > Molecular Epidemology Publications >

Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR.

Rajeevan MS, Vernon SD, Taysavang N, Unger ER.
Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR.
J Mol Diag 3:26-31, 2001.

Summary

Analysis of gene expression patterns from large numbers of specimens collected during epidemiologic studies is now feasible with the use of microarrays or high-density filter arrays. However, array results can be influenced by each step of the complex assay, from array manufacturing to sample preparation and image analysis. Array technology is most useful in establishing broad patterns of gene expression and in screening for differential gene expression. However, the field has not reached consensus on the magnitude of differences in gene expression levels that should be considered significant. Validation of differences in gene expression level is accomplished with an alternate method, most of which are not amenable to population-based studies of CFS. This manuscript explores the use of a kinetic real time-polymerase chain reaction (kinetic RT-PCR) to validate differentially expressed genes identified by high-density filter arrays. We used a human papillomavirus model system to test our validation approach. The kinetic RT-PCR was robust enough to validate relative changes in the expression of several genes present in varying amounts.

Abstract

We evaluated real-time (kinetic) reverse transcription-polymerase chain reaction (RT-PCR) to validate differentially expressed genes identified by DNA arrays. Gene expression of two keratinocyte subclones differing in the physical state of human papillomavirus (episomal or integrated) was used as a model system. High-density filter arrays identified 444 of 588 genes as either negative or expressed with less than twofold difference, and the other 144 genes as expressed uniquely or with more than twofold difference between the two subclones. Real-time RT-PCR used LightCycler-based SYBR Green I dye detection and melting curve analysis to validate the relative change in gene expression. Real-time RT-PCR confirmed the change in expression of 17 of 24 (71%) genes identified by high-density filter arrays. Genes with strong hybridization signals and at least twofold difference were likely to be validated by real-time RT-PCR. This data suggests that (i) both hybridization intensity and the level of differential expression determine the likelihood of validating high-density filter array results and (ii) genes identified by DNA arrays with a two- to fourfold difference in expression cannot be eliminated as false nor be accepted as true without validation. Real-time RT-PCR based on LightCycler technology is well-suited to validate DNA array results because it is quantitative, rapid, and requires 1000-fold less RNA than conventional assays.

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