Patch-Clamp Recording and RT-PCR on Single Cells

The technique described in this chapter provides electrophysiologists using patch-clamp with a convenient method to link electrophysiological data to a molecular analysis of the mRNAs expressed in a single cell. This molecular analysis can be used either to correlate cell responses with their molecular basis or to identify cell types according to the expression of specific markers. The core of the molecular analysis is polymerase chain reaction (PCR), which makes it fast, sensitive, and simple. Figure 1 outlines the general procedure followed in the experiments. Briefly, after recording of a cell with a patchclamp electrode, the cell content is aspirated through the tip of the electrode and expelled into a test tube with the whole content of the patch electrode. Reagents are then added to perform first strand cDNA synthesis from the mRNA present in the cell. After completion of the reverse transcription (RT) reaction, further reagents are added to the tube to enable a PCR reaction to amplify the cDNA(s) under investigation. Basically, one tube corresponds to one cell, since no change of tubes and very few biochemical manipulations are required. After the first PCR the amplified DNA is analyzed on agarose gel electrophoresis. In some instances, the DNA product from the first PCR is reamplified through a second