5.2. Using scAIR Positive Control RNA

The scAIR Positive Control RNA is designed for troubleshooting and measuring the sensitivity of the scAIR assay protocol. To run the positive control, aliquot 1 μl of scAIR positive control RNA in each well of a new 96-well plate with pre-aliquoted 5 μl of Hybridization Master mix. Freeze and store the plate at -80°C prior to use for the scAIR profiling experiment. For scAIR profiling, follow the standard protocol described below for experimental sorted T/B cells.

The scAIR Positive Control RNA included in the human scAIR kits is RNAare RNAs purified from CD3+ T cells or CD19+ B cells isolated from donor PBMC using CD3 or /CD19 antibody-magnetic beads. The scAIR Positive Control RNA included in the mouse and hybrid human/mouse scAIR kits is RNA purified from mouse spleen (CD1 strain) tissue.

For quality control of flow sorting and RNA integrity, we recommend depositing approximately 100 cells (in 1-2 μl of 1xPBS buffer) in one of the wells of the plate (with pre-aliquoted 5 μl of Hybridization Master mix). If scAIR NGS data analysis demonstrates efficient amplification of TCR/BCR clonotypes from positive pre-sort cell control but not from sorted cells, it indicates a problem with sorting of cells or degradation of RNA during the sorting process.