Flow Cytometric Analysis of Reverse Transcription-PCR Products: Quantification of p21WAF1/CIP1 and Proliferating Cell Nuclear Antigen mRNA

Background: Reverse transcription-PCR (RT-PCR) is a powerful tool in clinical diagnostics for analyzing even small amounts of RNA, but sensitive assays for quantifying the amplification products are time-consuming or expensive. Here we describe a novel flow cytometry-based assay for rapid and sensitive determination of relative amounts of RT-PCR products. Methods: For flow cytometric quantification, PCR products were labeled with both digoxigenin and biotin during amplification. Subsequently, amplicons were simultaneously bound to anti-digoxigenin microparticles and fluorescently labeled with streptavidin-R-phycoerythrin. Fluorescence intensity per bead was determined by flow cytometry. To study this assay, we examined the expression of the p21WAF1/CIP1 gene and the proliferating cell nuclear antigen (PCNA) gene in ultraviolet irradiation-exposed human keratinocytes lacking functional p53. Results: Fluorescence was linear with 60–10 000 pg of PCR product. As little as 0.4 fmol (40 pg of a 163-bp amplicon) of PCR product could be distinguished from background. The between-run CV of the fluorescent signal for 10 ng of p21 cDNA was 12% (n = 10). The fluorescence-template curve was sigmoidal. p21WAF1/CIP1 mRNA was decreased after ultraviolet irradiation of keratinocytes, whereas PCNA mRNA was markedly increased. Conclusion: The flow cytometric assay permits rapid (25 min) and reproducible identification of changes in mRNA abundance.