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. High-throughput analysis of gene expression is now feasible with the use of cDNA microarrays and high-density filter arrays (HDFA). However, array results can be influenced by each step of the complex assay, from array manufacturing to sample preparation (extraction, labeling, hybridization) and image analysis. [1][2][3] The efficiency of the reverse transcription (RT) reaction is known to be affected by the enzyme, primers, nucleotides, and RNA secondary structure. These factors in turn influence the representation of low-abundance transcripts in the final cDNA probe. 4,5 Complex cDNA probes can cross-hybridize to related sequences, and low-intensity hybridization signals are difficult to interpret. The field has not reached consensus on the significance of differences in hybridization intensity. Whereas some investigators interpret a twofold difference in hybridization intensity as evidence of differential gene expression, others require fourfold differences. 1,6,7 Currently, array technology is most useful in establishing broad patterns of gene expression and in screening for differential gene expression. Validation of expression differences is accomplished with an alternate method such as Northern blot hybridization or RNase protection assay. However, these assays are time-consuming, labor-intensive, and require large amounts of RNA (Ͼ5 g total RNA). Conventional reverse transcription-polymerase chain reaction (RT-PCR) can be done with smaller amounts of RNA (20 -40 ng), but quantification is difficult and relies on endpoint analysis of the PCR product. 8 -10 Real-time (kinetic) PCR evaluates product accumulation during the log-linear phase of the reaction and is currently the most accurate and reproducible approach to gene quantification. 9,10 In this study, we explored the applicability of kinetic RT-PCR as a rapid procedure for the validation of a number of differentially expressed genes identified by HDFA. Because of our interest in the interaction of human papillomavirus (HPV) on cellular gene expression, we used the HDFA expression profiles of two subclones differing in the integration status of HPV (integrated or mixed episomal/integrated) as a model system to test our validation approach. We found that a two-step RT-PCR using SYBR Green I dye detection with product verification by melting curve analysis is rapid, quantitative, and applicable to samples with limited amount of RNA. The method was robust enough to validate relative changes in the expression of a num...

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