An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells.

Exploring the variability in gene expressions of rare cells at the single-cell level is critical for understanding mechanisms of differentiation in tissue function and development as well as for disease diagnostics and cancer treatment. Such studies, however, have been hindered by major difficulties in tracking the identity of individual cells. We present an approach that combines single-cell picking, lysing, reverse transcription and digital polymerase chain reaction to enable the isolation, tracking and gene expression analysis of rare cells. The approach utilizes a photocleavage bead-based microfluidic device to synthesize and deliver stable cDNA for downstream gene expression analysis, thereby allowing chip-based integration of multiple reactions and facilitating the minimization of sample loss or contamination. The utility of the approach was demonstrated with QuantStudio digital PCR by analyzing the radiation and bystander effect on individual IMR90 human lung fibroblasts. Expression levels of the Cyclin-dependent kinase inhibitor 1a (CDKN1A), Growth/differentiation factor 15 (GDF15), and Prostaglandin-endoperoxide synthase 2 (PTGS2) genes, previously shown to have different responses to direct and bystander irradiation, were measured across individual control, microbeam-irradiated or bystander IMR90 cells. In addition to the confirmation of accurate tracking of cell treatments through the system and efficient analysis of single-cell responses, the results enable comparison of activation levels of different genes and provide insight into signaling pathways within individual cells.