Isolation and quantification of messenger RNA from tissue models by using a double-barrel carbon probe

摘要

In this study, we introduce the double-barrel carbon probe (DBCP)—a simple, affordable microring electrode— which enables the collection and analysis of single cells independent of cellular positioning. The target cells were punctured by utilizing an electric pulse between the two electrodes in DBCP, and the cellular lysates were collected by manual aspiration using the DBCP. The mRNA in the collected lysate was evaluated quantitatively using real-time PCR. The histograms of single-cell relative gene expression normalized to GAPDH were fit to a theoretical lognormal distribution. In the tissue culture model, we focused on angiogenesis to prove that multiple gene expression analysis was available. Finally, we applied DBCP for the embryonic stem (ES) cell-derived cardiomyocytes to substantiate the capability of the probe to collect cells, even from highvolume samples such as spheroids. Thismethod achieves high sensitivity for mRNA at the single-cell level and is applicable in the analysis of various biological samples independent of cellular positioning.