Quantitative RNA-polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced fluorescence.
Quantitative RNA-polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced fluorescence.
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定量 RNA-聚合酶链反应-通过毛细管电泳和激光诱导荧光进行 DNA 分析。
DOI：
10.1006/abio.1995.1019
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Kaminsky,LS
中科院分区：
文献类型：
--
作者：
Fasco,MJ;Treanor,CP;Spivack,S;Figge,HL;Kaminsky,LS
定量RNA聚合酶链反应（RNA-PCR）是一种非常强大的分析工具，由于其特异性和高水平的灵敏度。然而，定量RNA-PCR是高度劳动密集型的。目前还没有一种分析方法可以准确、快速地定量RNA-PCR反应混合物中的少量DNA。我们建立了一种利用毛细管电泳和激光诱导荧光检测PCR产生的DNA YOYO-1复合物的方法。RNA-PCR混合物可以直接分析（无需去除引物和蛋白质）或通过脱盐后的电动注射进行分析。在一系列混合物中测试了甘油醛-3-磷酸脱氢酶和P4501 A1的改良竞争性和多重竞争性RNA-PCR测定，所述混合物含有来自未处理的（基本上没有P4501 A1 mRNA）和2，3，7，8-四氯二苯并-p-二恶英处理的（高水平的P4501 A1 mRNA）HepG 2细胞的等浓度但不同比例的RNA。通过竞争性RNA-PCR可以检测到两种P4501 A1 mRNA溶液之间浓度的两倍差异。甘油醛-3-磷酸脱氢酶浓度始终恒定。多重竞争性PCR由于污染峰的存在而产生更多可变的结果，这阻碍了准确的面积积分。这些数据表明了毛细管电泳在各种定量PCR应用中的潜在有用性。
Quantitative RNA-polymerase chain reaction (RNA-PCR) is an extremely powerful analytical tool owing to its specificity and high level of sensitivity. Quantitative RNA-PCR is, however, highly labor intensive. No analytical method currently exists that can accurately and rapidly quantitate the small quantities of DNA in RNA-PCR reaction mixtures. We have developed a method using capillary electrophoresis and laser-induced fluorescence to detect YOYO-1 complexes of DNA produced by PCR. RNA-PCR mixtures can be analyzed either directly (without primer and protein removal) or by electrokinetic injection following desalting. Modified competitive and multiplex competitive RNA-PCR assays for glyceraldehyde-3-phosphate dehydrogenase and P4501A1 were tested in a series of mixtures containing equal concentrations, but different proportions, of RNA from untreated (essentially no P4501A1 mRNA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated (high levels of P4501A1 mRNA) HepG2 cells. Twofold differences in concentrations between two P4501A1 mRNA solutions could be detected by competitive RNA-PCR. Glyceraldehyde-3-phosphate dehydrogenase concentrations were constant throughout. Multiplex competitive PCR produced more variable results due to the presence of contaminating peaks, which hindered accurate area integration. These data demonstrate the potential usefulness of capillary electrophoresis in a variety of quantitative PCR applications.