Quantification of murine cytokine mRNAs using real time quantitative Reverse Transcriptase PCR
Quantification of murine cytokine mRNAs using real time quantitative Reverse Transcriptase PCR
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DOI：
10.1006/cyto.1998.0426
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发表时间：
1999-04-01
期刊：
影响因子：
3.8
通讯作者：
Mathieu, C
中科院分区：
文献类型：
--
作者：
Overbergh, L;Valckx, D;Mathieu, C
最近，一种新的“实时”定量逆转录酶-聚合酶链式反应技术被开发出来，它使用双标记的荧光探针来测量聚合酶链式反应的反应阶段的聚合酶产物的积累。这种方法可以通过在聚合酶链式反应过程中连续测量荧光发射的增加来直接检测聚合酶链式反应产物的形成。在这里，我们提供了用于定量检测小鼠细胞因子和其他免疫调节因子(IL-1、IL-2、IL-3、IL-5、IL-6、IL-7、IL-10、IL-12p40、IL-13、IL-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。此外，还构建了相应的参考质粒克隆VAS，允许对看家基因β-肌动蛋白或GAPDH进行直接定量和归一化。该分析非常灵敏和准确。这是一种“闭管”的聚合酶链式反应，避免了耗时和危险的聚合酶链式反应后操作，并降低了聚合酶链式反应污染的潜在风险。
Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the esponential 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-3, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gamma, TNF-alpha, TGF-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,vas 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. (C) 1999 Academic Press.