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Quantitative single-cell RT-PCR and Ca2+ imaging in brain slices

  • Cell and Molecular Physiology
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Abstract

We have established a quantitative reverse transcriptase-PCR (RT-PCR) approach for the analysis of RNA transcript levels in individual cells of living brain slices. Quantification is achieved by using rapid-cycle, real-time PCR protocols and high-resolution external cDNA standard curves for the gene of interest. The method consists of several procedures, including cell soma harvest, reverse transcription, and an optimized cDNA purification step, which allowed us to quantify transcripts in small types of neurons, like cerebellar granule cells. Thus, we detected in single granule cells an average of 20 transcript copies of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase. We combined two-photon calcium imaging and quantitative RT-PCR in single Purkinje and granule cells, respectively, and identified distinct glutamate receptor-dependent Ca2+ responses in these two cell types. The approach was further tested by profiling the expression of the ionotropic glutamate receptor subunits NR2B and NR2C in the cerebellum. Our study revealed a developmental switch from an average of 15 NR2B copies/cell at postnatal day 8 (P8) to about five NR2C copies/cell after P26. Taken together, our results demonstrate that the new method is rapid, highly sensitive, provides reliable results in neurons of various sizes, and can be used in combination with Ca2+ imaging.

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Acknowledgments

We thank Dr. Alexandra Lepier and Dr. John Davis for comments on earlier versions of the manuscript and I. Mühlhahn for technical assistance. The work was supported by grants from the DFG and the BMBF. Competing interests statement: The authors declare that they have no competing financial interests. This study was not financially supported by the companies selling reagents or instrumentation used in this approach.

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Authors and Affiliations

  1. Institut für Physiologie, Ludwig-Maximilians-Universität, Pettenkofer Straβe 12, 80336, Műnchen, Germany

    Guylaine M. Durand, Nima Marandi, Simone D. Herberger, Robert Blum & Arthur Konnerth

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  1. Guylaine M. Durand
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  2. Nima Marandi
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  3. Simone D. Herberger
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  4. Robert Blum
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  5. Arthur Konnerth
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Corresponding author

Correspondence to Robert Blum.

Additional information

Guylaine M. Durand and Nima Marandi contributed equally to this work.

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Durand, G.M., Marandi, N., Herberger, S.D. et al. Quantitative single-cell RT-PCR and Ca2+ imaging in brain slices. Pflugers Arch - Eur J Physiol 451, 716–726 (2006). https://doi.org/10.1007/s00424-005-1514-3

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  • DOI: https://doi.org/10.1007/s00424-005-1514-3

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