1.1. DriverMap™ AIR RNA Profiling

The DriverMap™ AIR RNA profiling assay is a next-generation targeted, multiplex RT-PCR technology designed for bulk expression profiling of all CDR3 TCR (TRA, TRB, TRD, TRG) and/or BCR (IGH, IGK, and IGL) clonotypes in a single reaction using RNA as the starting material. The assay is based on 315 forward primers designed for the FR3 variable region and 19 reverse primers designed for the conservative C-region of TCR/BCR mRNAs. These primers have been experimentally validated and optimized for the best performance. An additional set of primers is designed for the FR1 region, which allows (in combination with C region primers) the user to amplify full-length CDR1-CDR2-CDR3 V regions of all seven immune receptor genes (Fig. 1).

Fig 1. mRNA structure for TCR (alpha, beta, gamma, delta) and BCR (heavy, kappa, lambda) chains, and positions of forward and reverse PCR primers to amplify the CDR3 regions or CDR1-CDR2-CDR3 regions in AIR RNA assay.

The AIR RNA assay profiles all functional or productive isoforms and excludes non-functional pseudogenes and ORFs (as defined by the IMGT database https://www.imgt.org/IMGTrepertoire/) (Fig. 2). The AIR RNA assay is designed to profile the TCR and BCR repertoire together (preferred strategy) or separately (if necessary).

Fig. 2. Outline of DriverMap Multiplex RT-PCR Technology. Step 1: Reverse gene-specific primers (GSP) with UMI targeting all TCR and BCR C-region isoforms, are hybridized with mRNA and extended with reverse transcriptase. Step 2: Forward GSPs are annealed with cDNA template and extended by DNA polymerase. Step 3: In the first PCR step, anchored universal primers (AP1 and AP2) are amplified CDR3 (or CDR1-CDR2-CDR3) cDNA fragments. Step 4: Second PCR amplifies CDR3 fragments using indexed primers. The indexed amplified products are then analyzed by NGS.