Introduction

Fluorescence-activated cell sorting (FACS) is a branch of flow cytometry that allows for isolation of specific cell populations that can then be further evaluated by Next Generation Sequencing. When utilizing FACS for population isolation prior to NextGen sequencing, it is important to consider the protection of RNA from RNase activity, environmental conditions, and the sort efficiency to ensure optimum sample quality.

Methods

Canine Myeloid Derived Suppressor Cells (MDSC) were isolated from peripheral blood samples of healthy canines and oral melanoma patients. Multiple techniques were utilized for the protection of RNA prior to, during, and after FACS. After ideal RNA quality was obtained, single-cell analysis using the 10x Genomics platform was performed on a population of sorted cells from a healthy canine and a melanoma patient.

Results

A combination of RNAlater™ and an RNase inhibitor (RiboLock™) was found to provide high-quality RNA from FACS sorted canine MDSCs. Enrichment of the rare cell population also improved the sorting efficiency and dramatically reduced the time needed for sorting. Single-cell analysis was carried out and provided good quality RNA that is currently undergoing further analysis.

Conclusion

Overall, there are multiple techniques that can be used to protect RNA prior to NextGen sequencing and a combination of these techniques is recommended to provide the best information from samples while minimizing gene changes due to stress and degradation. As shown here, accounting for protection of RNA allowed for isolation of good quality RNA that allowed for sample evaluation at a single cell level.

Funding Information

This work was supported by a resident research grant from the University of Florida, College of Veterinary Medicine and donated funding from the Olive’s Way Foundation and the Milner Comparative Oncology Lab.