Kurokawa M, Furukawa H, Yabe T, Matsui T, Toda M, Hamada C, Kasukawa R, Yamamoto K, Nishioka K, Kato T
Rheumatology, Immunology and Genetic Program, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Japan.
J Immunol Methods. 1999 Apr 22;224(1-2):203-8. doi: 10.1016/s0022-1759(99)00022-8.
In analyses of antigen-specific immune responses, it is essential to estimate the frequency of individual T cell clonotypes. This frequency has been estimated, however, only indirectly by the frequency of T cell receptor (TCR) mRNA. We have developed a method to determine T cell frequency directly by cell count using reverse transcription polymerase chain reaction (RT-PCR) amplification of TCR beta genes from single cell-derived cDNA (single cell PCR). In a study of clinical samples, the frequency of clonally expanded T cells estimated by TCR frequency analysis was found to be higher than that by single cell PCR. Single cell PCR can estimate T cell frequency accurately, as it is not affected by skewed PCR amplification or different TCR mRNA expressions in individual T cells.
