Premixed Controls
| Observation | Possible Cause | Recommended Action |
|---|---|---|
| Detected cDNA molecule numbers for Spike-in controls are significantly higher (e.g.,>5x) than cDNA molecule numbers for most abundant RNA/DNA clonotypes. | Low content or high diversity of immune receptor RNA/DNA genes in the sample, RNA/DNA is degraded or has a wrong concentration. | Reduce the amount of Spike-in control mix added to experimental RNA/DNA samples. Measure RNA/DNA concentration, re-purify, replace or use more amount of RNA/DNA samples. |
| Abundant Spike-in control cDNA molecule numbers are less than 100, low yield of amplified products for both experimental and positive control samples. | Possible manual error in the protocol. | Add extra cycles in 2nd PCR step, and repeat the AIR protocol using positive control RNA/DNA. |
| Abundant Spike-in control cDNA molecule numbers are less than 100 for experimental samples but higher (e.g., 500-1,000) for positive control RNA/DNA samples. | Presence of inhibitors in RNA samples | Add extra cycles in 2nd PCR for experimental samples, use alternative source, or re-purify RNA/DNA samples |
| Lower than expected 16:4:1 ratio in cDNA molecules for different Spike-in controls. | PCR overcycling, background in measurement of AIR control constructs, and not enough NGS reads. | Reduce PCR cycle number (in 1st PCR), use control RNA without Spike-in controls to measure Spike-in control background level, replace AIR reagents, use PCR-free area/conditions to re-run AIR assay, use separate test tubes rather than plate to minimize cross-contamination level, run NGS with more reads per sample, optimize parameters for reduction background level based on ration reads per cDNA molecule. |
| Lower reproducibility in cDNA molecule number between different samples or in triplicates. | Not equal concentration of Spike-in controls, Spike-In RNA controls are not fully dissolved (RNA molecules could “stick” to each other), not equal primer extension/amplification conditions for different samples. | Heat Spike-in controls at 70°C for 2 min and pipet the whole volume several (e.g., 10) times before use, optimize liquid handling/PCR conditions to have an equal yield of PCR products in triplicate samples and use master mixes for common reagents. |
Triplex Isoform Pools
| Observation | Possible Cause | Recommended Action |
|---|---|---|
| Low level of cross-contamination between different experimental samples | <0.1% or less cross-contamination | Based on our experience, a level of cross-contamination less than or equal to 0.1% is commonly detected due to the high sensitivity of AIR assay |
| High level of cross-contamination between different experimental samples | >1% cross-contamination | Mistakes in AIR assay conditions or sample handling. Use PCR-free area/conditions to re-run AIR assay, use separate test tubes rather than plate to minimize cross-contamination level, replace AIR reagents, and use control RNA without added Spike-in control as a “negative” control to monitor the level of sample cross-contamination. |
Last modified:
24 February 2025
Need more help with this?
Contact Us