Overbergh L, Valckx D, Waer M, Mathieu C
Laboratory for Experimental Transplantation, U.Z.Gasthuisberg, Catholic University of Leuven, Herestraat 49, Leuven, 3000, Belgium.
Cytokine. 1999 Apr;11(4):305-12. doi: 10.1006/cyto.1998.0426.
Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.
最近，一种用于“实时”定量逆转录聚合酶链反应的新技术已经开发出来，该技术使用双标记荧光探针在聚合酶链反应的指数期测量聚合酶链反应产物的积累。这种方法通过在聚合酶链反应过程中连续测量荧光发射的增加，直接检测聚合酶链反应产物的形成。在此，我们展示了验证这种聚合酶链反应方法用于定量小鼠细胞因子和其他在免疫调节中起作用的因子（白细胞介素-1、白细胞介素-2、白细胞介素-4、白细胞介素-5、白细胞介素-6、白细胞介素-7、白细胞介素-10、白细胞介素-12p40、白细胞介素-13、白细胞介素-15、干扰素-γ、肿瘤坏死因子-α、转化生长因子-β和诱导型一氧化氮合酶）的数据。对于每种感兴趣的物质，设计了一组引物和内部探针，它们特异性地扩增目标互补脱氧核糖核酸，而不共扩增污染的基因组脱氧核糖核酸。此外，构建了相应的参考质粒互补脱氧核糖核酸克隆，允许直接定量。另外，对管家基因β-肌动蛋白或甘油醛-3-磷酸脱氢酶进行了标准化。该检测方法非常灵敏和准确。它是一种“闭管”聚合酶链反应，避免了耗时且危险的聚合酶链反应后操作，并降低了聚合酶链反应污染的潜在风险。
