Single-neuron RNA-Seq: technical feasibility and reproducibility
- 1Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA
- 2Department of Cell and Neurobiology, University of Southern California, Los Angeles, CA, USA
- 3Illumina, Inc, Hayward, CA, USA
- 4Department of Psychiatry, University of Southern California, Los Angeles, CA, USA
Understanding brain function involves improved knowledge about how the genome specifies such a large diversity of neuronal types. Transcriptome analysis of single neurons has been previously described using gene expression microarrays. Using high-throughput transcriptome sequencing (RNA-Seq), we have developed a method to perform single-neuron RNA-Seq. Following electrophysiology recording from an individual neuron, total RNA was extracted by aspirating the cellular contents into a fine glass electrode tip. The mRNAs were reverse transcribed and amplified to construct a single-neuron cDNA library, and subsequently subjected to high-throughput sequencing. This approach was applied to both individual neurons cultured from embryonic mouse hippocampus, as well as neocortical neurons from live brain slices. We found that the average pairwise Spearman’s rank correlation coefficient of gene expression level expressed as RPKM (reads per kilobase of transcript per million mapped reads) was 0.51 between five cultured neuronal cells, whereas the same measure between three cortical layer 5 neurons in situ was 0.25. The data suggest that there may be greater heterogeneity of the cortical neurons, as compared to neurons in vitro. The results demonstrate the technical feasibility and reproducibility of RNA-Seq in capturing a part of the transcriptome landscape of single neurons, and confirmed that morphologically identical neurons, even from the same region, have distinct gene expression patterns.
Keywords: cell culture, electrophysiology, gene expression, neuron, RNA-Seq, transcriptome
Citation: Qiu S, Luo S, Evgrafov O, Li R, Schroth GP, Levitt P, Knowles JA and Wang K, (2012) Single-neuron RNA-Seq: technical feasibility and reproducibility. Front. Gene. 3:124. doi: 10.3389/fgene.2012.00124
Received: 14 April 2012; Accepted: 19 June 2012;
Published online: 06 July 2012.
Copyright: © 2012 Qiu, Luo, Evgrafov, Li, Schroth, Levitt, Knowles and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.
*Correspondence: James A. Knowles and Kai Wang, Department of Psychiatry, University of Southern California, Health Sciences Campus, ZNI 401, M/C 2821, Los Angeles, CA 90089-2821, USA. e-mail: firstname.lastname@example.org; email@example.com