Gey A, Hamdi S, Vielh P, Mehtali M, Fridman W H, Tartour E
Unité d'Immunologie Clinique, INSERM U 255, Institut Curie, Paris, France.
J Immunol Methods. 1999 Jul 30;227(1-2):149-60. doi: 10.1016/s0022-1759(99)00078-2.
We have developed an original protocol of direct in situ RT-PCR with biotinylated labeled primers to detect cytokine mRNA inside cells. This label improved the specificity of the technique compared with the use of digoxigenin or fluorescein-labeled primers. We found a reliable correlation between the known expression of cytokine mRNA in a given cell and a positive signal with in situ RT-PCR. Nuclear counterstaining demonstrated that the positive signal obtained was distributed in the cytoplasm in accordance with mRNA localization. In addition, direct demonstration of the presence of the expected PCR product in cell extracts without non-specific parasitic DNA amplification provided strong support for the specificity of the method. Designing the primers in order to prevent DNA amplification, the use of recombinant Thermus thermophilus (rTth) DNA polymerase and a decreased duration of each cycle of PCR by combining the annealing and hybridization steps improved the reproducibility and reliability of the technique and morphological preservation of the cells. Experiments in which different proportions of cytokine mRNA positive and negative cells were mixed argue against significant diffusion of PCR product into initially cytokine mRNA negative cells, thereby leading to false-positive results. In comparison with the direct incorporation of labeled dNTP during amplification, our procedure appears to ensure greater specificity and does not need DNAse treatment which is often difficult to standardize. Detection of IL-2 and IFNgamma mRNA induction after T cell activation using this direct in situ RT-PCR method showed that the technique may be helpful for monitoring cytokine gene expression at a single cell level.
我们开发了一种使用生物素化标记引物的原位直接逆转录聚合酶链反应（RT-PCR）原始方案，用于检测细胞内的细胞因子信使核糖核酸（mRNA）。与使用地高辛配基或荧光素标记引物相比，这种标记提高了该技术的特异性。我们发现给定细胞中已知的细胞因子mRNA表达与原位RT-PCR的阳性信号之间存在可靠的相关性。细胞核复染表明，获得的阳性信号根据mRNA定位分布在细胞质中。此外，在细胞提取物中直接证明存在预期的PCR产物且无非特异性寄生DNA扩增，为该方法的特异性提供了有力支持。通过设计引物以防止DNA扩增、使用重组嗜热栖热菌（rTth）DNA聚合酶以及通过合并退火和杂交步骤缩短每个PCR循环的持续时间，提高了该技术的可重复性和可靠性以及细胞的形态保存。将不同比例的细胞因子mRNA阳性和阴性细胞混合的实验表明，PCR产物不会大量扩散到最初细胞因子mRNA阴性的细胞中，从而导致假阳性结果。与在扩增过程中直接掺入标记的脱氧核苷三磷酸（dNTP）相比，我们的方法似乎确保了更高的特异性，并且不需要经常难以标准化的脱氧核糖核酸酶（DNAse）处理。使用这种原位直接RT-PCR方法检测T细胞活化后白细胞介素-2（IL-2）和γ干扰素（IFNγ）mRNA的诱导情况表明，该技术可能有助于在单细胞水平监测细胞因子基因表达。
