Quantitative PCR for detection of the OT-1 transgene

We show excellent correlation between flow cytometric assessment of OT-1 cells and OT-1 signal by qPCR assays in cell dilutions as well as in in vivo adoptive transfer experiments. We also demonstrate that qPCR can be performed from archival formalin-fixed paraffin-embedded tissue sections. In addition, the non-quantitative PCR using the OT-1-specific primers without the real-time probe is a valuable tool for OT-1 genotyping, obviating the need for peripheral blood collection and subsequent flow cytometric analysis.An OT-1 specific qPCR assay has been developed to quantify adoptively transferred OT-1 cells. OT-1 qPCR to determine cell signal is a valuable adjunct to the standard flow cytometric analysis of OT-1 cell number, particularly in experimental settings where tissue disaggregation is not desirable or in tissues which are not readily disassociatedAdoptive transfer of CD8+ T cells from MHC Class I-restricted OVA specific T cell receptor (TCR) transgenic mice (OT-1) into host C57BL/6 mice allows for the activated CD8+ T cell population to be tracked and quantified after stimulation with the OVA peptide SIINFEKL [1] using standard flow cytometric analysis. Using this methodology, it has been demonstrated that activated CD8+ T cells undergo migration to the liver where they are trapped and undergo apoptosis [2]. While flow cytometry is sensitive and specific, it is best suited to tissues from which single cell suspensions can be obtained easily, such as the spleen and peripheral lymph nodes. Although flow cytometry can be performed on lymphocytes isolated from other tissues such as liver, muscle and intestine, it is technically more challenging and questions may be raised regarding whether the cell sample is representative of the in vivo state. Reports by Lang (1997), Lim (2002), and Gallard (2002), [3-5] also emphasize these points and describe PCR based approaches for tracking specific T-cell clonotypes. Additionally, sample preparation for histologic analysis