Wedemeyer N, Göhde W, Pötter T
Department of Radiobiology, Westfälische Wilhelms-Universität Münster, Robert-Koch-Strasse 43, 48149 Münster, Germany. Author for correspondence. Fax 49-251-8355303, USA.
Clin Chem. 2000 Aug;46(8 Pt 1):1057-64.
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.
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.
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.
The flow cytometric assay permits rapid (25 min) and reproducible identification of changes in mRNA abundance.
逆转录聚合酶链反应（RT-PCR）是临床诊断中用于分析少量RNA的强大工具，但用于定量扩增产物的灵敏检测方法耗时或昂贵。在此，我们描述了一种基于流式细胞术的新型检测方法，用于快速、灵敏地测定RT-PCR产物的相对量。
为了进行流式细胞术定量，PCR产物在扩增过程中用洋地黄毒苷和生物素进行标记。随后，扩增子同时与抗洋地黄毒苷微粒结合，并用链霉亲和素-R-藻红蛋白进行荧光标记。通过流式细胞术测定每个微珠的荧光强度。为了研究该检测方法，我们检测了缺乏功能性p53的紫外线照射的人角质形成细胞中p21（WAF1/CIP1）基因和增殖细胞核抗原（PCNA）基因的表达。
荧光与60 - 10000 pg的PCR产物呈线性关系。低至0.4 fmol（40 pg的163 bp扩增子）的PCR产物可与背景区分开来。10 ng p21 cDNA荧光信号的批间变异系数为12%（n = 10）。荧光-模板曲线呈S形。角质形成细胞紫外线照射后p21（WAF1/CIP1）mRNA减少，而PCNA mRNA显著增加。
流式细胞术检测可快速（25分钟）且可重复地鉴定mRNA丰度的变化。
