[Analysis of multiple cytokine mRNA at a single cell level]

To analyze multiple cytokine expression in individual cells, we have developed a new RT-PCR method. At the first step, a FACS sorted single cell was lysed by detergent and cDNA was synthesized immediately using an oligo dT primer. Then cDNA was amplified by PCR using 15-base oligonucleotide random primers. These procedures were done in a single well of a microtiter-plate without transfer of samples to avoid the loss of mRNA. The second PCR was performed from a small aliquot of the amplified cDNA to detect the specific gene expression. Preamplification of cDNA enabled analysis of more than 50 species of mRNA expression from a single cell. This technique will be a very powerful tool to examine small numbers of cells purified from autoimmune infiltrates, etc.