A synthetic standard DNA construct for use in quantification of murine cytokine mRNA molecules.
A synthetic standard DNA construct for use in quantification of murine cytokine mRNA molecules.
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用于定量鼠细胞因子 mRNA 分子的合成标准 DNA 构建体。
DOI：
10.1016/0161-5890(95)00061-i
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发表时间：
1995
影响因子：
3.6
通讯作者：
Street,NE
中科院分区：
文献类型：
--
作者：
Farrar,JD;Street,NE
已经开发了合成DNA构建体作为标准分子，由此可以通过逆转录-聚合酶链反应（RT-PCR）来定量鼠细胞因子mRNA分子。命名为Cytoquant 1的构建体允许定量鼠IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-10、IFN-γ、TNF-α、TGF-β、GM-CSF、CD 4、CD 8、HPRT和β-肌动蛋白mRNA水平。该技术基于扩增Cytoquant 1转录的RNA分子作为RT和PCR反应中的内标对照。来自这些分析的定量数据以绝对值表示，即分子/细胞，这允许比较来自单独实验的数据。在这项研究中，已经在有和没有刺激的Th 1和Th 2细胞克隆中定量了编码β-肌动蛋白、IL-10、IFN-γ和GM-CSF的mRNA。定量分析数据是高度可重复的，并且细胞因子mRNA浓度反映了受限的细胞因子分泌模式。此外，组成型细胞因子mRNA水平在静息细胞中是可检测的，消除了对外源刺激的需要。高度的灵敏度和准确性使得该方法独特地适合于在体内和体外生物模型中研究T细胞亚群细胞因子表达。
A synthetic DNA construct has been developed as a standard molecule whereby murine cytokine mRNA molecules can be quantified by the reverse transcription-polymerase chain reaction (RT-PCR). The construct, designated Cytoquant 1, allows the quantification of murine IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-γ, TNF-α, TGF-β, GM-CSF, CD4, CD8, HPRT and β-actin mRNA levels. This technique is based on the amplification of a transcribed RNA molecule from Cytoquant 1 as an internal standard control in both the RT and PCR reactions. The quantification data from these analyses are expressed in absolute values, i.e. molecules/cell, which allows the data derived from separate experiments to be compared. In this study, mRNAs encoding β-actin, IL-10, IFN-γ and GM-CSF have been quantitated in both Th1 and Th2 cell clones with, and without, stimulation. The quantitative analysis data are highly reproducible and cytokine mRNA concentrations are reflective of restricted cytokine secretion patterns. Furthermore, constitutive cytokine mRNA levels are detectable in resting cells, eliminating the need for exogenous stimulation. The high degree of sensitivity and accuracy make this methodology uniquely suited for the study of T-cell subset cytokine expression in both in vivo and in vitro biological models.