Digital PCR to determine the number of transcripts from single neurons after patch-clamp recording
Nóra Faragó,
Ágnes K. Kocsis,
Sándor Lovas,
Gábor Molnár,
Eszter Boldog,
Márton Rózsa,
Viktor Szemenyei,
Enikő Vámos,
Lajos I. Nagy,
Gábor Tamás,
László G. Puskás
Affiliations
Nóra Faragó
1Avidin Ltd, Szeged, Hungary
Ágnes K. Kocsis
3Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szege, Hungary
Sándor Lovas
3Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szege, Hungary
Gábor Molnár
3Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szege, Hungary
Eszter Boldog
3Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szege, Hungary
Márton Rózsa
3Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szege, Hungary
Viktor Szemenyei
3Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szege, Hungary
Enikő Vámos
3Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szege, Hungary
Lajos I. Nagy
1Avidin Ltd, Szeged, Hungary
Gábor Tamás
3Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szege, Hungary
Whole-cell patch-clamp recording enables detection of electrophysiological signals from single neurons as well as harvesting of perisomatic RNA through the patch pipette for subsequent gene expression analysis. Amplification and profiling of RNA with traditional quantitative real-time PCR (qRT-PCR) do not provide exact quantitation due to experimental variation caused by the limited amount of nucleic acid in a single cell. Here we describe a protocol for quantifying mRNA or miRNA expression in individual neurons after patch-clamp recording using high-density nanocapillary digital PCR (dPCR). Expression of a known cell-type dependent marker gene (gabrd), as well as oxidative-stress related induction of hspb1 and hmox1 expression, was quantified in individual neurogliaform and pyramidal cells, respectively. The miRNA mir-132, which plays a role in neurodevelopment, was found to be equally expressed in three different types of neurons. The accuracy and sensitivity of this method were further validated using synthetic spike-in templates and by detecting genes with very low levels of expression.
