Rare Transcripts Quantification by Strand-Specific RT-qPCR

B lymphocytes development is associated with significant immunoglobulin (Ig) gene recombination leading to the establishment of a highly diverse and specific Ig repertoire. Accessibility of Ig genes is tightly regulated and associated with the production of sterile transcripts (noncoding), including low abundant anti-sense (AS) transcripts. Initially described by Corcoran's lab, the method used to quantify such transcripts at Ig loci is based on strand-specific reverse transcription (RT) from RNA and can be applied to any other cell types. The detection of strand-specific transcripts requires specific experimental controls run in order to exclude false signals that could arise from RT random priming. This chapter details the different steps of the whole procedure, from RNA isolation (optimized to a small number of cells) and multiplex RT reaction to cDNA amplification, including experimental controls.