Question: Why is the annealing temperature during Pre-Amplification updated from 67°C to 63°C across all Fixed RNA Profiling protocols?
- The temperature change was made in order to improve Feature Barcode sensitivity and yield when utilizing the Barcode Oligo Capture protocols (Feature Barcoding with TotalSeq™️-C).
- There is improvement in Gene Expression and Feature Barcode library sensitivity/complexity when performing Pre-Amplification with the reduced temperature of on-market products.
- Because there were benefits in improved assay performance across the portfolio, this update was needed for the most optimal results with Barcode Oligo Capture, and having one Pre-Amplification PCR setting would streamline user experience we incorporated this change across all protocols.
Figure 1. Singleplex Gene (left) and TotalSeq™️-B Protein (right) library sensitivity/complexity when performing Pre-Amplification with an annealing temperature at 67°C (previously recommended temperature) and 63°C (newly recommended temperature). Quality metrics and overall cell calling are unaffected by the change but sensitivity is improved when using the new temperature.
Please note: the Sample Index PCR recommended cycles have been updated due to the increased yield from Pre-Amplification when implementing this change. Refer to the most up-to-date User Guide for details.
See the following Q&A articles for additional details:
- What happens if I forget to update my thermal cycler program for Pre-Amplification and I perform Barcode Oligo Capture protocols?
- When should I start to incorporate the thermal cycler protocol adjustment (updating Pre-Amplification annealing temperature)?
Product: Fixed RNA Profiling, Single Cell Gene Expression Flex