Flow cytometric analysis of reverse transcription-PCR products: quantification of p21(WAF1/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 p21(WAF1/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. p21(WAF1/CIP1) mRNA was decreased after ultraviolet irradiation of keratinocytes, whereas PCNA mRNA was markedly increased.


CONCLUSIONS
The flow cytometric assay permits rapid (25 min) and reproducible identification of changes in mRNA abundance.
Department of Radiobiology, Westfälische Wilhelms-Universität Münster, Robert-Koch-Strasse 43, 48149 Münster, Germany. Author for correspondence. Fax 49-251-8355303, USA.
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Wedemeyer
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Göhde
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Pötter
D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000032:analysis* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018809:Proliferating Cell Nuclear Antigen