Quantifying Gene Expression Directly from FACS Using Hydrolysis (TaqMan) Probes on Flash-frozen Cells

Background

This protocol is a modification and experimental validation of the TaqMan Gene Expression Cells-to-Ct^TM Kit User Guide, Publication Number 4385117 Revision E (2012) (Thermo Fisher Scientific/Life Technologies) for flash-frozen, rather than fresh, cells to allow for transportation among core facility laboratories (Procedure B). Using fresh cells requires all steps of the process to be performed sequentially with no stopping point at one facility. Using flash-frozen cells allows the separation of the cell sorting and downstream steps at different locations while ensuring the viability of the cells. The cell sorting is unique to this protocol. It is important to maintain the viability of the cells to ensure the accurate measurement of cellular genomes and transcriptomes (Gawad et al., 2016). Flash-freezing also provides a stopping point for sorting the cells and the downstream analysis. This protocol is also designed and validated for use with a ‘homebrew’ qPCR master mix allowing the analysis of gene expression from a larger number of samples and/or assays than originally allocated by the manufacture’s kit.

We attempt to consistently use the nomenclature recommended by the MIQE guidelines for qPCR (Bustin et al., 2009). Thermo Fisher Scientific uses Cts (cycles at threshold), and their kit is named as such, but the recommended standardization is Cqs (quantification cycles). ‘TaqMan’ probes/assays are standardized as hydrolysis probes.