6.1.2. RNA

Starting Amount of RNA:

The table below provides a general guideline for starting amount of RNA for AIR TCR/BCR profiling assay:

• The optimal range for AIR assay is 25-100 ng of total RNA from a large number of immune cells e.g., PBMC, buffy coat, whole blood, purified immune cell fractions (more than 50,000), or lymphoid tissues with a high percentage of immune cells. For PBMC RNA we recommend using 50 ng, and for whole blood RNA, we recommend 100 ng.

• Although the standard AIR protocol works reliably in the 1 ng-1,000 ng range, for deep, quantitative AIR repertoire profiling, we recommend using 25-100 ng of total RNA (from whole blood, PBMC samples, or isolated immune cells). Using larger amounts of total RNA can improve the detection of rare clonotypes, but it requires increasing the sequencing depth (and NGS cost) which can affect expression levels of high-medium abundant clonotypes. Using smaller amounts of total RNA will reduce the number of clonotypes that can be reliably profiled. For example, running 5-10 ng of total whole blood/PBMC RNA instead of 50 ng will profile ~200-500 highly abundant clonotypes (i.e., transcripts with >10 target CDR mRNA molecules) as opposed to ~1,000 clonotypes.

• For RNA isolated from tissue samples with a low percentage of immune cells (e.g., cancer biopsies, non-lymphoid tissues), the amount should be increased to use 200-1,000 ng of total RNA. Due to the high specificity of the AIR assay, the total RNA can be used directly without purification of immune cell fraction.