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RACE-PCR
Rapid Amplification of cDNA Ends (RACE)-PCR combined with switch-oligo nested PCR is a robust approach for TCR and BCR analysis on RNA samples. It provides the advantages of amplification-based methods without bringing in multiplex PCR-related biases.
Coupling high sensitivity and accurate quantification
RACE-PCR relies on a switch-oligo approach to place an adapter at the 5’ region. This allows the use of a single primer instead of a panel targeting the multiple V regions. cDNA is specifically synthesized from TCR or BCR molecules and subsequently amplified during nested PCR steps to simultaneously prepare the DNA fragments for high-throughput sequencing. We optimized this protocol to maximize specificity and sensitivity while maintaining time efficiency. Our dedicated bioinformatics pipelines provide insights into immune repertoire diversity, dynamics, and clonality, without compromising on quantification accuracy, thanks to the unique molecular identifiers.
Reduced biases compared to multiplex PCR
RACE-PCR does not require primers hybridizing to the V regions thus overcoming the main disadvantages of multiplex PCR – the current most widely used technology for immune repertoire analysis. In multiplex PCR, a mix of reverse primers located on the V regions is used. This hampers the detection of novel exons and V allele variants and multiplies the PCR bias introduced, thus distorting the relative abundances of the different immune cell populations.
RACE-PCR enables a highly sensitive, cost- and time-effective immune repertoire analysis, particularly suitable when cells numbers or RNA material are limited. It constitutes a powerful immunopharmacogenomics tool enabling immune repertoire diversity analysis to the investigation of drug responses.
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