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 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.