A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells.
BACKGROUND
Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation.


RESULTS
We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR.


CONCLUSIONS
Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses.
Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ, USA ; School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA.
Jordan R
JR
Yaron
Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ, USA.
Colleen P
CP
Ziegler
Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ, USA.
Thai H
TH
Tran
Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ, USA.
Honor L
HL
Glenn
Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ, USA.
Deirdre R
DR
Meldrum
Fluorescence microscopy; Gene expression analysis; RT-qPCR; Single cell