ANALYSIS OF CYTOKINE MESSENGER-RNA AND DNA - DETECTION AND QUANTITATION BY COMPETITIVE POLYMERASE CHAIN-REACTION
ANALYSIS OF CYTOKINE MESSENGER-RNA AND DNA - DETECTION AND QUANTITATION BY COMPETITIVE POLYMERASE CHAIN-REACTION
复制标题
DOI：
10.1073/pnas.87.7.2725
复制
发表时间：
1990-04-01
影响因子：
11.1
通讯作者：
BUNN, HF
中科院分区：
文献类型：
--
作者：
GILLILAND, G;PERRIN, S;BUNN, HF
两种细胞因子，粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素3（IL-3）的表达，已在MLA-144细胞诱导前后与佛波醇12-肉豆蔻酸酯13-乙酸酯进行了研究。我们描述了一个适应的聚合酶链反应（PCR）的mRNA或DNA从少量的细胞进行高度准确的定量。将含有待测RNA的cDNA拷贝的PCR混合物的等分试样加入到竞争DNA片段的系列稀释液中，所述竞争DNA片段通过具有小内含子或突变的内部限制性内切酶位点而不同于感兴趣的cDNA。因此，使用相同的引物共扩增未知和竞争产品。产物的比率在扩增过程中保持恒定，并且可以容易地定量。在未刺激的细胞中，不能检测到GM-CSF或IL-3 mRNA。然而，在适当的诱导下，在少至200个细胞中检测并定量两种细胞因子的mRNA。竞争性PCR还用于准确定量正常人细胞、5 q综合征患者细胞克隆群和已知仅具有一个人GM-CSF基因拷贝的人仓鼠细胞系中人GM-CSF基因的拷贝数。
The expression of two cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3), has been investigated in MLA-144 cells before and after induction with phorbol 12-myristate 13-acetate. We describe an adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells. Aliquots of the PCR mixture containing cDNA copies of the RNA to be assayed were added to serial dilutions of a competitor DNA fragment that differed from the cDNA of interest by having either a small intron or a mutated internal restriction enzyme site. Therefore, the same primers were used to coamplify the unknown and the competitor. The ratio of products remains constant through the amplification and can be readily quantitated. In unstimulated cells, no GM-CSF or IL-3 mRNA could be detected. However, with appropriate induction, mRNA for both cytokines was detected and quantitated in as few as 200 cells. Competitive PCR was also used to accurately quantitate the copy number of the human GM-CSF gene in normal human cells, in a clonal population of cells from a patient with 5q- syndrome, and in a human-hamster cell line known to have only one copy of the human GM-CSF gene.