The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 μm above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10 μs between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after the electric pulse, less than 1 μL of the lysed solution was collected using a microinjector followed by RNA purification and first strand cDNA synthesis. Real-time PCR was performed to quantify the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression inside the single cell. The average copy numbers of GAPDH mRNA collected by the electrical cell lysis method were found to be comparable to those obtained by a simple capillary suction method. Although single-cell analysis has already been demonstrated, we have shown for the first time that the fast electrical cell lysis can be used for quantitative mRNA analysis at the single-cell level. This electrical cell lysis method was further applied for the analysis of mRNA obtained from single spheroids - the aggregated cellular masses formed during the three-dimensional culture - as a model system to isolate small cellular clusters from tissues and organs.
ASJC Scopus subject areas
- Analytical Chemistry