RT-PCR strategies used in the detection of clonotypic IgH transcripts.

At the top of the figure is a schematic diagram of the structure of the immunoglobulin heavy chain gene, including the Vh leader, complementarity determining regions (CDRs), and constant chains (μ, δ, γ, α, and ε). Primers specific to unique CDR2 and CDR3 sequences in the MM clone were generated. All strategies were performed using cDNA derived from patient or mouse samples. Strategy A involved 35 cycles of PCR with CDR3 and constant chain (μ, δ, γ, and α) primers. Strategy B involved 35 cycles of PCR with primers to Vh leader and a given constant chain, followed by reamplification with 25 cycles of PCR using patient-specific CDR3 and nested constant chain primers. Strategy C involved 35 cycles of PCR with primers to the Vh leader and a constant chain to amplify only the relevant isotype in each reaction, independent of its VDJ rearrangement. All samples were also subjected to a single-stage RT-PCR using patient-specific CDR2/CDR3 primers (strategy D).