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Digital PCR to determine the number of transcripts from single neurons aſter patch-clamp recording Nóra Faragó1,2,*

, Ágnes K. Kocsis3* Szemenyei3 , Enikő Vámos3 1Avidin Ltd., Szeged, Hungary, 2 , Sándor Lovas3 , Lajos I. Nagy1 , Gábor Molnár3 , Gábor Tamás3 Center, Hungarian Academy of Sciences, Szeged, Hungary, and 3

BioTechniques 54:327-336 (June 2013) doi 10.2144/000114029 Keywords: Digital PCR; single cell PCR; patch-clamp recording; gene expression

Supplementary material for this article is available at *

N.F. and A.K.K contributed equally to this work.

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 indi- vidual 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 indi- vidual 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.

Gene expression analyses typically require RNA from thousands of cells, but neu- rons of specific types cannot be isolated in such large quantities. Population-average expression profiling of multiple neurons provides an incomplete picture, mask- ing transcription patterns of individual cells that reflect physiological roles in the brain, activation state, or disease pheno- type in a complex microcircuit (1–2). In this context, transcription profiling from whole brain tissue or populations of neu- rons isolated by fluorescence activated cell sorting (FACS) with specific antibodies cannot fully relate expression changes to a certain cell type or distinguish cell spe- cific patterns (3–4). Tis is also the case

Method summary:

We present an approach for determining exact mRNA or miRNA copy numbers in single neurons after patch-clamp recording by using high-density nanocapillary digital PCR technology. This method can identify individual genes participating in the establishment and maintenance of particular neuronal phenotypes, deconvolve different neuronal cell types, and determine the exact distribution or variability of gene expression profiles for electrophysiologically phenotyped cells more precisely than classical single cell qPCR.

Vol. 54 | No. 6 | 2013 327

for laser capture microdissection where the selection criteria are based on cellular morphology (5). To determine physiological relation-

ships within complex neuronal microen- vironments in single, live neurons, whole-cell patch-clamp recording is an optimal approach. When coupled with gene expression analysis from the same cell, it provides a better understanding of how changes at the molecular level are manifested at the level of cell function and also enables better classification of different neuron types (6–7). Aſter whole-cell patch- clamp recording of electrophysiological signals from single neurons, RNA from the perisomatic region can be harvested

through the patch pipette. Linear (6) or exponential (PCR based) (7) amplification techniques can then be used to analyze expression of individual genes. Lambolez and colleagues were the first to combine patch-clamp recordings with PCR-based gene expression analysis of single neurons in culture (7). Using targeted pream- plification and subsequent quantitative real-time PCR (qRT-PCR), one can detect tens or hundreds of mRNA or miRNA molecules(8). In principle, this approach can be also applied to RNA harvested from patch-clamped cells. When faced with sensitivity concerns

related to single cell qRT-PCR, digital PCR (dPCR) is a logical alternative (9–11). In

, Eszter Boldog3 , and László G. Puskás1,2

Laboratory for Functional Genomics, Department of Genetics, Biological Research Research Group for Cortical Microcircuits of the

Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szeged, Hungary , Márton Rózsa3 , Viktor

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