Distinct Kinetics in Electrophoretic Extraction of Cytoplasmic RNA from Single Cells

The physical fractionation of cytoplasmic versus nuclear
components
of cells is a key step for studying the subcellular localization of
molecules. The application of an electric field is an emerging method
for subcellular fractionation of proteins and nucleic acids from single
cells. However, the multibiophysical process that involves electrical
lysis of cytoplasmic membranes, electrophoresis, and diffusion of
charged molecules remains unclear. Here we study RNA dynamics in single
cells during the electrophoretic extraction via a microfluidic system
that enables stringent fractionation of the subcellular components
leveraging a focused electric field. We identified two distinct kinetics
in the extraction of RNA molecules, which were respectively associated
with soluble RNA and mitochondrial RNA. We show that the extraction
kinetics of soluble RNA is dominated by electrophoresis over diffusion
and has a time constant of 0.15 s. Interestingly, the extraction of
mitochondrial RNA showed unexpected heterogeneity in the extraction
with slower kinetics (3.8 s), while reproducibly resulting in
the extraction of 98.9% ± 2% after 40 s. Together, we uncover
that the microfluidic system uniquely offers length bias-free fractionation
of RNA molecules for quantitative analysis of correlations among subcellular
compartments by exploiting the homogeneous electrophoretic properties
of RNA.