3.2. Profiling Samples with Low Amount of Lymphocyte

When working with a small number of lymphocyte cells, such as cancer biopsy, non-lymphoid tissue, biological fluid, tissue or blood microsamples and FFPE samples, we recommend purifying RNA from at least 1 mg of the tissue sample. For bulk tissue with a small number of lymphocytes (e.g., 100-10,000), we recommend using as much RNA as possible (up to 1,000-2,000 ng of total RNA) to capture most of the receptor diversity (most abundant clonotypes) (Fig.12). Using a lower input amount will reduce the detection efficiency of medium-abundant clonotypes. For the AIR DNA assay, lymphocyte DNA will be diluted by DNA from other cell types, and as a result, the detection sensitivity of TCR and BCR clonotypes will be significantly compromised. If possible, please consider purifying the lymphocyte cell fraction prior to DNA isolation to increase the sensitivity of the AIR DNA assay.

If the size of the tissue sample is less than 1 mg (e.g., micro-dissected tumor samples or FFPE slide) or using immune fractions purified by FACS or antibody-magnetic beads from samples with lower lymphocyte counts (e.g., tissue, blood, fluid, or tumor cells), we recommend using the DirectCell™ Protocol to directly profile immune receptors from cell lysate without purifying RNA or DNA. Based on our experience, direct profiling of isolated immune cells increases the detection sensitivity of the AIR assay by 5- to 10-fold, which can be explained by avoiding the loss of RNA/DNA during purification steps. Due to the lower complexity of TCR/BCR clonotypes in these samples, we recommend using approximately 2 million reads per sample (or at least 20 reads per UMI).

Fig. 15.Reproducibility of AIR RNA assay in profiling top 100 TRB clonotypes in 20 ng of PBMC RNA mixed with up to 50-fold excess total RNA from mouse brain.