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Clinical Chemistry 46: 1057-1064, 2000;
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(Clinical Chemistry. 2000;46:1057-1064.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

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

Niels Wedemeyera,1, Wolfgang Göhde1 and Thomas Pötter1

1 Department of Radiobiology, Westfälische Wilhelms-Universität Münster, Robert-Koch-Strasse 43, 48149 Münster, Germany.
a Author for correspondence. Fax 49-251-8355303; e-mail wedemey{at}uni-muenster.de

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.




The following articles in journals at HighWire Press have cited this article:


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 Molecular Cancer TherapeuticsHome page
T. Hata, H. Yamamoto, C. Y. Ngan, M. Koi, A. Takagi, B. Damdinsuren, M. Yasui, Y. Fujie, T. Matsuzaki, H. Hemmi, et al.
Role of p21waf1/cip1 in effects of oxaliplatin in colorectal cancer cells
Mol. Cancer Ther., October 1, 2005; 4(10): 1585 - 1594.
[Abstract] [Full Text] [PDF]

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 Clin. Chem.Home page
N. Wedemeyer, T. Potter, S. Wetzlich, and W. Gohde
Flow Cytometric Quantification of Competitive Reverse Transcription-PCR Products
Clin. Chem., September 1, 2002; 48(9): 1398 - 1405.
[Abstract] [Full Text] [PDF]


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