6.3.1.1. Positive Control Experiment

Prior to running experimental samples, we strongly recommend that you run a positive control experiment with a set of T/B cells (e.g., from any non-experimental PBMC sample) with the positive control RNA included in the kit. The positive control experiment is designed to troubleshoot potential problems in the sorting step, to check the stability of RNA in sorted cells, and to measure the sensitivity of the scAIR assay protocol.

To run the positive control experiment, take one of the PCR plates with pre-aliquoted 5 µl of Hybridization Master Mixes (prepared in Step 5.1), and deposit the following:
1. 100 presorted T/B cells into 24 wells (e.g., A1-H3). Dilute presorted cells (e.g., PBMC) in 1xPBS to a concentration of approximately 4 T/B cells/µl based on the estimated percent of T/B cells in the presort sample and aliquot 1 µl per well.
2. 100 T/B cells sorted into 24 wells (four cells per well, e.g., A4-H6).
3. 25 T/B cells sorted into 24 wells (one cell per well, e.g., A7-H9).
4. Aliquot 1 μl of scAIR positive control RNA provided in the kit in 24 wells (e.g., A10-H12).

Freeze and store the positive control plate at -80°C before use for the scAIR profiling experiment. For scAIR profiling using a positive control plate:
1. Follow the standard protocol described below (Step 6) but run each subset of presorted, sorted, and positive control RNA as a separate sample by combining together hybridization reactions (after adding SPRIselect beads, step 6.1.5) from each subset of 24 wells.
2. Analyze yield of amplified cDNA products after 2nd amplification step (with indexed primers) as described in Section 7.1.
If analysis of the amplified indexed libraries reveals amplification of expected PCR products (Fig.3), you could proceed with scAIR profiling of your experimental samples.
3. If you can see amplification products in presorted and positive control RNA samples, but not in sorted cells, it could indicate problems with FACS gating, sorting, or degradation of RNA in sorted cells. Based on our experience, degradation of RNA in sorted cells is a common problem that could be solved by using the Cell Sorter Cleaning Best Practices as described in the paper. If no amplification products are seen in sorted cells, but amplification products are present in control RNA and pre-sorted cells, please test the presence of RNAse in the fluid collected after sorting by using the RNaseAlert™ Lab Test Kit.