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dilution spectrum can be explained by the presence of more than 1 template molecule per nanoliter volume reaction. Terefore, if more than 30 cDNA copies per sample are present in 1 cell, the samples should be diluted or evenly distributed among 512 or more individual subreactions.


Figure 2. Firing pattern and microscopic reconstructions. (A) Firing pattern and (B) light microscopic reconstructions of a neurogliaform cell (left), a fast spiking basket cell (middle) and a pyramidal cell (right). Somata and dendrites are shown in red and axons in black.


mRNA expression analysis of single neurons with dPCR With a similar experimental setup (see the flowchart of the dPCR protocol in Supple- mentary Figure S1), we were able to detect individual genes from single neurons aſter patch-clamp recording. Te cellular content of an individual neuron was aspirated into the recording pipette and expelled directly into a low-adsorbtion test tube containing first strand cDNA synthesis buffer with RNase inhibitor to prevent RNA degradation. RT reactions were performed with RT primers for the gene of interest and extracts from individual cells. Subsequently, the RT reaction mixture, TaqMan probes, and qRT-PCR reagents were mixed and evenly distributed among the 256 (4 subarrays) nanocapillary holes on an OpenArray plate. Te expression of slc2a4 (GLUT4) was


analyzed in individual neurogliaform cells aſter patch-clamp recording and was found to have 12–25 (18.75 ± 5.38) copies per cell (n = 4) (Figure 1B). In order to demonstrate reproducibility and sensitivity, 3 independent RT reactions were prepared with single cell aspirates and approximately similar numbers of copies (24 molecules) of the synthetic spike-in rps18 templates (s_rps18) compared to the estimated number of slc2a4 messages per cell (reaction mixtures: RT#1-RT#3). One-third of the RT mixture was applied to dPCR amplifi- cation of slc2a4 and one-third from the same RT mixture to detect the number of s_rps18 molecules (n = 3). Digital images as well as individual positive amplification reactions are shown in Figure 1B. An unexpectedly even representation of the s_rps18 molecules (12, 13, and 13 copies) could be recorded in the 3 independent experiments, validating the reproducibility of our protocol. Moreover, when multiplying the identified slc2a4 cDNA molecules by 3, we found an almost perfect match (17 ± 3.46, n = 3) with the results obtained from the non-diluted samples.


Figure 3. mRNA and miRNA expression in different neuron types. Expression of rps18, gabrd, and mir-132 in neurogliaform cells (NGFC), fast-spiking basket cells (FSC), and pyramidal cells (PC). No template controls (negc) from the cerebral cortex were determined by single cell QRT-PCR or dPCR.


Vol. 54 | No. 6 | 2013 333


Cell-type specific mRNA expression analysis of single neurons with dPCR In order to obtain cell-type specific mRNA expression data with our protocol, three exten- sively characterized cell types from the supra- granular layers of the rat somatosensory cortex were selected. Whole cell recorded neuro- gliaform cells , fast spiking basket cells, and pyramidal cells were identified based on their late spiking, fast spiking, and regular spiking


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