Figure 4

Dilutional series of lymphoma DNA analyzed by conventional and real-time PCR. Lymphoma samples of a dog with B cell lymphoma (A) and T cell lymphoma (B) were serially diluted into regular lymphoid DNA (rlDNA; 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%) and amplified with IgH major and TCRγ primer sets. The minimal concentration producing a visible band was 3.13% and 12.5% for B and T cell clonal rearrangement with conventional PCR and subsequent PAGE. The detection limit for real-time PCR with MCA was 1.5% and 6.25% for B and T cell clonal rearrangement, based on the cut-off of a –d(F1)/dT ratio from sample DNA to regular lymphoid DNA of at least 1.5:1 for IgH major and 2:1 for TCRγ. The regular lymphoid DNA was prepared as a mix of 12 lymph node samples of 3 dogs to adjust for peak height variations between individual samples as observed for the amplification of regular lymphoid DNA with the TCRγ primer set (Figure 2B). Gel images include one lane with 100% lymphoma DNA and a non-template control (NTC).