Strategies for profiling single mouse intestinal epithelial cells by targeted gene expression
McDowell, W., Box, A., Staehling, K. et al.
Targeted gene expression profiling of single cells permits the study of heterogeneity in cell populations. Here, a pool of mouse intestinal crypt-base CD44+/GRP78- cells was collected by fluorescence activated cell sorting. Aliquots were either loaded onto the Fluidigm® C1™ System for microfluidic cell capture and cDNA synthesis in nanoliter volumes, or flow-sorted directly into individual PCR plate wells for cDNA synthesis in microliter volumes. The pre-amplified cDNAs were transferred to the BioMark™ System for EvaGreen® real-time PCR. The two sample preparation methods were compared by expression analysis of 86 genes, using Fluidigm's SINGuLAR™ R-scripts. After outlier identification, gene expression values from 42% of the “C1” and 92% of the “flow” wells were retained. For 55 of the genes, expression was measured in both the “C1” and “flow” cells. Genes with a high variance in expression likely stemming from the sample preparation method and/or unspecific amplification were removed. Hierarchical clustering on the remaining data revealed gene clusters that contributed to the expected Lgr5hi and Lgr5lo intestinal stem cell (ISC) populations as well as a small population of differentiated cells. The subpopulations could be defined by either method. However, as ISCs quickly undergo apoptosis at room temperature, the use of the C1 System provided no clear advantage over the direct sorting of the fragile cells into lysis/RT reaction buffer. Specifically, the C1 quality control step to verify the number of captured cells and cell viability was omitted to accelerate processing.
McDowell, W., Box, A., Staehling, K. et al. "Strategies for profiling single mouse intestinal epithelial cells by targeted gene expression" Journal of Biomolecular Techniques (2014): S26–7