1. DriverMap™ AIR RNA Profiling Assay

The DriverMap™ AIR TCR-BCR Profiling Kit combines multiplexed RT-PCR amplification with the depth and precision of Next-Generation Sequencing (NGS) to quantitatively measure gene expression and analyze RNA sequences of all functional immune receptor mRNA isoforms for TCR chains (TRA, TRB, TRD, TRG) and BCR chains (IGH, IGK, and IGL) with the exclusion of non-functional pseudogenes and ORFs (as defined by IMGT database http://www.imgt.org/IMGTrepertoire/).

The AIR assay allows profiling of the repertoire of antigen-binding CDR3 regions using a set of experimentally validated forward primers (designed for variable FR3 region), and reverse primers (designed for the conserved C region) of TCR and BCR mRNAs. Two sets of AIR primers specific for TCR (TRA, TRB, TRD, and TRG) and BCR (IGH, IGL, and IGK) chains are provided separately in AIR reagent kits to allow profiling AIR repertoires individually for TCRs or BCRs or together in a single reaction.

Main advantages of DriverMap™ AIR Assay:

• An easy-to-run, one-tube, single-day assay allows profiling of target transcripts from total RNA isolated from whole blood, PBMC, tissue, or directly from a small number (e.g., 100-50,000) of purified immune cells without prior RNA isolation.
• The assay provides robust, quantitative, and reproducible measurements of the copy number of clonotype mRNAs over as many as 4-orders of magnitude differences in expression level.
• To assay provides increased sensitivity from a small amount of RNA (1-50 ng), the DriverMap protocol employs several mechanisms to minimize amplification of off-target and primer dimer background products.
• No mRNA enrichment, rRNA, mitochondrial, beta-globin depletion, or other processing required.
• The primers are designed using Illumina’s DNA/RNA UD Indexes labeling strategy to minimize NGS index-swapping background issues and amplified products are compatible with 300-n paired-end NGS sequencing using Illumina’s NextSeq/NovaSeq instruments.
• The reverse gene-specific primers are designed with Unique Molecular Identifiers (UMIs), which facilitates the accurate quantification of the copy number of cDNA molecules used in amplification steps and detection of low abundance clonotypes above background level.
• NGS data can be analyzed with the MiXCR software package available online. Cellecta also provides custom data analysis services on request.

Fig. 1. Outline of DriverMap AIR RT-PCR-NGS assay for expression and sequence analysis of immune receptor mRNA regions

Additional Profiling Assays Compatible with the AIR RNA Assay:

• DriverMap™ AIR TCR/BCR Profiling Kit (human DNA) allows comprehensive CDR3 repertoire analysis from gDNA for all four TCR chains (TRA, TRB, TRD, and TRG) and/or three BCR chains (IGH, IGK, and IGL) using primers designed for FR3 and J regions. Although AIR RNA provides higher sensitivity than AIR DNA assay, both assays could be effectively used together for the identification of antigen-induced clonotypes. For more details, please refer to AIR Technical Guide.
• DriverMap™ EXP T/B500 Immune Marker Profiling Kit is designed for deep characterization of T and B immune cell fractions (e.g., naïve, effector, memory, exhausted, etc.) using a set of primers designed for 500 highly informative T-cell and B-cell subtyping and activation marker genes. Combined profiling of AIR repertoire and T/B marker genes in isolated (e.g., by FACS or magnetic beads) immune cell fractions provide a comprehensive phenotypic characterization of T/B cell subtypes associated with different TCR/BCR clonotypes.
• DriverMap™ EXP Genome-Wide Profiling Kit is a convenient single-tube RT-PCR-NGS protocol that robustly measures the expression level of all 19,000 human protein-coding genes starting from a small amount of total RNA (down to single-cell level). Combined EXP and AIR profiling can be used for comprehensive phenotypic characterization of RNA samples and unbiased discovery of novel biomarkers associated with specific CDR3 clonotypes.

Related Products and Services:

• AIR profiling assay in whole blood microsamples. AIR repertoire analysis could be performed without RNA purification directly in 30 µl of whole blood collected, stabilized, and dried using microsampling technology tips from Neoteryx.
  

For more detailed recommendations related to the design of your experiment, please refer to the AIR Guide brochure. For more information on Cellecta’s DriverMap kits and services, please visit the DriverMap Adaptive Immune Receptor (AIR) Profiling Service webpage.

Please read the entire user manual before proceeding with your experiment.

*Click the ⤓ Download as PDF link located at the bottom of the left menu to download the PDF version of this user manual.

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