Recently, a new single-cell quantitative Reverse Transcriptase (qRT)-PCR approach for the determination of RNA transcript levels in brain slice preparations has been described¹. However, it could not distinguish between cells that did not express the mRNA of interest or when the cell harvest or the qPCR failed. In the course of analyzing expression levels of connexin 36 (Cx36) and pannexin 2 (Px2) in individual pyramidal neurons of the cortex, we developed an improved quantitative single-cell RT-PCR protocol. A modified cell-harvest solution, an optimized RT reaction and new priming strategies increased the cDNA templates up to 100-fold in comparison to the previous approach¹. Using random hexamers and gene-specific reverse primers allowed the quantitative analysis of expression levels of Cx36 or Px2 in comparison with the housekeeping gene GAPDH. A developmental expression analysis revealed that Cx36 or Px2 are expressed only in a subset of GAPDH-positive pyramidal neurons. Thus, quantitative GAPDH analysis as an internal control for cell harvest, RT-efficiency and single-cell cDNA purification results in improved quantitative single-cell RT-PCR for the analysis of complex gene-specific expression patterns in living neurons. Supported by DFG, Su 104/3-3 & Bl 567/2-2. 1Durand, Marandi, Herberger, Blum, & Konnerth. Pflügers Arch (2005).