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

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.

Figure 1.

Scatterplot of the log base 2 of the hybridization intensities for each gene in subclones 20863 and 20861 as determined by HDFA. Perpendicular lines (dashed) indicate the threshold values for each subclone. The two solid parallel lines indicate the position of twofold differences in intensity. Genes on and outside the parallel lines were identified as differentially expressed by HDFA.

Figure 2.

Fluorescence versus cycle number plot of G3PDH transcript in subclones 20863 and 20861 by LightCycler RT-PCR assay based on SYBR Green I dye detection. Both subclones were evaluated for relative levels of G3PDH transcript using 1:200, 1:2000, and 1:20,000 dilutions of cDNA which are indicated as A, B, and C, respectively, in the amplification plot. There were four reactions per dilution per sample. At each dilution, all reactions overlapped in the log-linear phase, indicating identical levels of G3PDH transcript in both samples. Closed arrowheads indicate no-RT reactions with both subclones. Open arrowhead shows PCR with no template. Striped arrow indicates the position of noise band. All data points below the noise band were excluded from quantification. Annealing and signal acquisition temperatures for G3PDH are given in Table 1 .