Abstract
This protocol details a method for analyzing the expression of multiple genes from a single Purkinje neuron, including the determination of whether the gene expression is monoallelic or biallelic. The protocol describes how to extract a single, living Purkinje cell for reverse transcription, divide the cDNAs into three equal samples and subject those to triplicate amplification of multiple targets by two rounds of PCR (first a multiplex PCR then a gene-specific nested PCR) and finally discriminate the allelic expression of the transcript by direct sequencing of the PCR products. In optimal conditions, this method permits the analysis of the expression of 18 genes in a single Purkinje cell. This protocol can be completed in 5–6 d.
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Acknowledgements
We thank H. Kato, M. Kawaguchi and K. Hirano for their assistance and discussion. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (Molecular Brain Science) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (14104025 to T.Y.), Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (17024034 to T.Y.) and CREST in the Japan Science and Technology Corporation (T.Y.).
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Esumi, S., Kaneko, R., Kawamura, Y. et al. Split single-cell RT-PCR analysis of Purkinje cells. Nat Protoc 1, 2143–2151 (2006). https://doi.org/10.1038/nprot.2006.343
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DOI: https://doi.org/10.1038/nprot.2006.343
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