Single Cell RT-PCR for the Detection of Cytokine mRNA
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The introduction of the polymerase chain reaction (PCR) methodology has dramatically revolutionized basic and clinical molecular biology research in the recent decade. Accurate detection and identification of tiny and limited amounts of DNA are now possible (1 ).
The PCR method, in combination with cell isolation and purification techniques has promoted the identification and characterization of specific mRNA types and the equivalent proteins produced in these single cells, which differ from those expressed by the other cells in their vicinity. Following this development, it was shortly demonstrated that this method is applicable and valid for identifying mRNA expressed by an individual cell (2 ).
Single cell reverse transcription (RT-PCR) is advantageous in assessing the pattern of a specific mRNA expression by cells that are widely distributed and intermingled with other tissue cells and are not easily identified by their morphology or histology.
We modified the single cell RT-PCR approach previously described (2 ) into a reproducible and friendly technique, for studying and characterization of certain biochemical events in mast cells cocultured with fibroblasts (3 ). This original work clearly showed enhancement of interleukin (IL)-3 accumulation in connective tissue mast cells cocultured with fibroblasts. This may explain the physiologic role of IL-3 in maintaining the survival of mast cell subpopulations and probably other cells.
