Dynamics of RNA in single cells under focused electric field

Abstract

Recently, we developed a microfluidic technique leveraging electrophoretic extraction of RNAs for stringent fractionation of cytoplasmic and nuclear RNAs from single cells [1-3]. To dissect the biophysics underlying the extraction, we explored the dynamics of RNA molecules using fluorescence microscopy and finite element analyses. The fluorescence visualization revealed distinct kinetics in the extraction of soluble RNA and mitochondrial RNA, which were respectively characterized by time constants of 0.15 s and 3.8 s (Fig.1A). Interestingly, the extraction of soluble RNA showed highly repeatable dynamics, while the mitochondrial RNA showed heterogeneous transient. To investigate the effect of the diffusivities of RNA molecules, we numerically solved the current conservation and the asymptotic Smoluchowski equations for cell lysis and the LaplaceNernst-Planck equations for RNA transport. Our numerical model successfully captured the extraction dynamics of soluble RNA molecules (Fig.1B). The model revealed that the extraction of soluble RNA molecules is dominated by electrophoresis over diffusion and the phenomenon is well characterized by dimensionless time (t* = tμE/a, μ is electrophoretic mobility, E is electric field inside the channel, and a is the cell diameter) and Peclet number (Pe = μEa/D, D is the diffusion coefficient). The duration of the extraction is almost constant over the three orders of magnitude of the Peclet number, except for the slightly elongated duration of highly diffusive molecules (Pe < 10⁴) (Inset of Fig.1B). By limiting the extraction time as 1 s, we found an enrichment of short RNA molecules (shorter than 650 nt) in the nuclear fractions in comparison to 40-s extraction, supporting the elongation of extraction with highly diffusive molecules. Our microfluidic technique uses 40-s extraction to ensure the uniform extraction of various RNA molecules for unbiased downstream analysis of gene expression.