Development of a direct in situ RT-PCR method using labeled primers to detect cytokine mRNA inside cells

We have developed an original protocol of direct in situ RT-PCR with biotin ylated labeled primers to detect cytokine mRNA inside cells. This label imp roved the specificity of the technique compared with the use of digoxigenin or fluorescein-labeled primers. We found a reliable correlation between th e known expression of cytokine mRNA in a given cell and a positive signal w ith in situ RT-PCR, Nuclear counterstaining demonstrated that the positive signal obtained was distributed in the cytoplasm in accordance with mRNA lo calization. In addition, direct demonstration of the presence of the expect ed PCR product in cell extracts without non-specific parasitic DNA amplific ation provided strong support for the specificity of the method. Designing the primers in order to prevent DNA amplification, the use of recombinant T hermus thermophilus (rTth) DNA polymerase and a decreased duration of each cycle of PCR by combining the annealing and hybridization steps improved th e reproducibility and reliability of the technique and morphological preser vation of the cells. Experiments in which different proportions of cytokine mRNA positive and negative cells were mixed argue against significant diff usion of PCR product into initially cytokine mRNA negative cells, thereby l eading to false-positive results. In comparison with the direct incorporati on of labeled dNTP during amplification, our procedure appears to ensure gr eater specificity and does not need DNAse treatment which is often difficul t to standardize. Detection of IL-2 and IFN gamma mRNA induction after T ce ll activation using this direct in situ RT-PCR method showed that the techn ique may be helpful for monitoring cytokine gene expression at a single cel l level. (C) 1999 Elsevier Science B.V. All rights reserved.