Gene Expression Analysis by Multiplex Single-Cell RT-PCR

  • Ludovic Tricoire
  • Bruno Cauli
  • Bertrand LambolezEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1941)


Brain circuit assemblies comprise different cellular subpopulations that exhibit morphological, electrophysiological, and molecular diversity. Here we describe a protocol which, combined with whole-cell patch-clamp recording and morphological reconstruction, allows the transcriptomic analysis of the recorded cell. This protocol provides recipes on how to detect simultaneously the expression of 24 genes/markers at the single-cell level using polymerase chain reaction (PCR), how to design gene-specific probes, and how to validate them. This technique provides multiplexed expression data that cannot be easily obtained by other approaches such as immunological co-labeling.

Key words

mRNA cDNA Cell type Transcriptome Electrophysiology 


  1. 1.
    Lambolez B, Audinat E, Bochet P, Crepel F, Rossier J (1992) Ampa receptor subunits expressed by single purkinje-cells. Neuron 9(2):247–258. CrossRefPubMedGoogle Scholar
  2. 2.
    Bochet P, Audinat E, Lambolez B, Crepel F, Rossier J, Iino M, Tsuzuki K, Ozawa S (1994) Subunit composition at the single-cell level explains functional-properties of a glutamate-gated channel. Neuron 12(2):383–388. CrossRefPubMedGoogle Scholar
  3. 3.
    Jonas P, Racca C, Sakmann B, Seeburg PH, Monyer H (1994) Differences in Ca2+ permeability of Ampa-type glutamate-receptor channels in neocortical neurons caused by differential Glur-B subunit expression. Neuron 12(6):1281–1289. CrossRefPubMedGoogle Scholar
  4. 4.
    Tsuzuki K, Lambolez B, Rossier J, Ozawa S (2001) Absolute quantification of AMPA receptor subunit mRNAs in single hippocampal neurons. J Neurochem 77(6):1650–1659. CrossRefPubMedGoogle Scholar
  5. 5.
    Lambolez B, Ropert N, Perrais D, Rossier J, Hestrin S (1996) Correlation between kinetics and RNA splicing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in neocortical neurons. Proc Natl Acad Sci USA 93(5):1797–1802. CrossRefPubMedGoogle Scholar
  6. 6.
    Angulo MC, Lambolez B, Audinat E, Hestrin S, Rossier J (1997) Subunit composition, kinetic, and permeation properties of AMPA receptors in single neocortical nonpyramidal cells. J Neurosci 17(17):6685–6696CrossRefGoogle Scholar
  7. 7.
    Hepp R, Hay YA, Aguado C, Lujan R, Dauphinot L, Potier MC, Nomura S, Poirel O, El Mestikawy S, Lambolez B, Tricoire L (2015) Glutamate receptors of the delta family are widely expressed in the adult brain. Brain Struct Funct 220(5):2797–2815. CrossRefPubMedGoogle Scholar
  8. 8.
    Liss B, Franz O, Sewing S, Bruns R, Neuhoff H, Roeper J (2001) Tuning pacemaker frequency of individual dopaminergic neurons by Kv4.3L and KChip3.1 transcription. EMBO J 20(20):5715–5724. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Tricoire L, Pelkey KA, Erkkila BE, Jeffries BW, Yuan XQ, McBain CJ (2011) A blueprint for the spatiotemporal origins of mouse hippocampal interneuron diversity. J Neurosci 31(30):10948–10970. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Cauli B, Audinat E, Lambolez B, Angulo MC, Ropert N, Tsuzuki K, Hestrin S, Rossier J (1997) Molecular and physiological diversity of cortical nonpyramidal cells. J Neurosci 17(10):3894–3906CrossRefGoogle Scholar
  11. 11.
    Karagiannis A, Gallopin T, David C, Battaglia D, Geoffroy H, Rossier J, Hillman EMC, Staiger JF, Cauli B (2009) Classification of NPY-expressing neocortical interneurons. J Neurosci 29(11):3642–3659. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Perrenoud Q, Rossier J, Geoffroy H, Vitalis T, Gallopin T (2013) Diversity of GABAergic interneurons in layer VIa and VIb of mouse barrel cortex. Cereb Cortex 23(2):423–441. CrossRefPubMedGoogle Scholar
  13. 13.
    Ferezou I, Cauli B, Hill EL, Rossier J, Hamel E, Lambolez B (2002) 5-HT3 receptors mediate serotonergic fast synaptic excitation of neocortical vasoactive intestinal peptide/cholecystokinin interneurons. J Neurosci 22(17):7389–7397CrossRefGoogle Scholar
  14. 14.
    Ferezou I, Hill EL, Cauli B, Gibelin N, Kaneko T, Rossier J, Lambolez B (2007) Extensive overlap of mu-opioid and nicotinic sensitivity in cortical interneurons. Cereb Cortex 17(8):1948–1957CrossRefGoogle Scholar
  15. 15.
    Hill EL, Gallopin T, Ferezou I, Cauli B, Rossier J, Schweitzer P, Lambolez B (2007) Functional CB1 receptors are broadly expressed in neocortical GABAergic and glutamatergic neurons. J Neurophysiol 97(4):2580–2589. CrossRefPubMedGoogle Scholar
  16. 16.
    Porter JT, Cauli B, Tsuzuki K, Lambolez B, Rossier J, Audinat E (1999) Selective excitation of subtypes of neocortical interneurons by nicotinic receptors. J Neurosci 19(13):5228–5235CrossRefGoogle Scholar
  17. 17.
    Battaglia D, Karagiannis A, Gallopin T, Gutch HW, Cauli B (2013) Beyond the frontiers of neuronal types. Front Neural Circuit 7. ARTN 13
  18. 18.
    Pohlkamp T, David C, Cauli B, Gallopin T, Bouche E, Karagiannis A, May P, Herz J, Frotscher M, Staiger JF, Bock HH (2014) Characterization and distribution of Reelin-positive interneuron subtypes in the rat barrel cortex. Cereb Cortex 24(11):3046–3058. CrossRefPubMedGoogle Scholar
  19. 19.
    Cadwell CR, Palasantza A, Jiang XL, Berens P, Deng QL, Yilmaz M, Reimer J, Shen S, Bethge M, Tolias KF, Sandberg R, Tolias AS (2016) Electrophysiological, transcriptomic and morphologic profiling of single neurons using patch-seq. Nat Biotechnol 34(2):199–203. CrossRefPubMedGoogle Scholar
  20. 20.
    Fuzik J, Zeisel A, Mate Z, Calvigioni D, Yanagawa Y, Szabo G, Linnarsson S, Harkany T (2016) Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes. Nat Biotechnol 34(2):175–183. CrossRefPubMedGoogle Scholar
  21. 21.
    Cauli B, Porter JT, Tsuzuki K, Lambolez B, Rossier J, Quenet B, Audinat E (2000) Classification of fusiform neocortical interneurons based on unsupervised clustering. Proc Natl Acad Sci USA 97(11):6144–6149. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Ludovic Tricoire
    • 1
  • Bruno Cauli
    • 1
  • Bertrand Lambolez
    • 1
    Email author
  1. 1.Sorbonne Université, UPMC Univ Paris 06 UM119, Centre National de la Recherche Scientifique (CNRS) UMR8246, Institut National de la Santé et de la Recherche Médicale (INSERM) UMRS1130, Neuroscience Paris Seine,Institut de Biologie Paris-SeineParisFrance

Personalised recommendations