Reports


dPCR, single cDNA molecules are amplified in subreactions to generate binary calls. When cDNA molecules are partitioned evenly among hundreds of individual reactions, an absolute readout of total DNA copy number can be obtained. By generating thousands of subre- actions on nanofluidic arrays, digital arrays represent the most powerful solution for high- throughput qRT-PCR and dPCR measure- ments (12–16). Here we present the first report of the


combination of dPCR, using the OpenArray platform (Life Technologies, Carlsbad, CA), and patch-clamp techniques to assess mRNA and miRNA expression in single neurons. Tis opens the door to sophisticated gene expression and regulatory network analysis on electrophys- iologically manipulated neuronal cells.


Materials and methods


Electrophysiology All procedures were conducted with the approval of the University of Szeged and in accordance with the National Institutes of Health Guide to the Care and Use of Laboratory Animals. Wistar male rats (P22–40) were anesthetized by intraperitoneal injection of ketamine (30 mg/kg) and xylazine (10 mg/kg). Following decapitation, coronal slices (350 µm thick) were prepared from the somatosensory cortex. Slices were incubated at room temperature for 1 h in artificial cerebrospinal fluid (130 mM NaCl, 3.5 mM KCl, 1 mM NaH2 NaHCO3


D(+)-glucose) saturated with 95% O2 CO2


, 1 mM CaCl, 3 mM MgSO4 PO4


differed only in that 3 mM CaCl2 MgSO4


, 24 mM , 10 mM and 5%


. Te solution used during recordings and 1.5 mM


were used (17). In an oxidative stress


model experiment, hydrogen peroxide (0.3 mM final concentration) was added to the artificial cerebrospinal fluid and applied to cortical slice preparations for 2 h. All other procedures were conducted with the same protocol. Micropi- pettes (5–7 MΩ) were filled with intracellular solution (126 mM K-gluconate, 4 mM KCl, 10 mM HEPES, 10 mM creatine phosphate, 8 mM biocytin; pH 7.25; 300 mOsm) supple- mented with RNase Inhibitor (1U/µl, Life Technologies) to prevent any RNA degradation. Somatic whole-cell recordings were obtained at ~36°C from neurogliaform cells, pyramidal cells, and fast spiking interneurons. Cells were visualized by infrared differential interference contrast videomicroscopy (Axioskop micro- scope, Carl Zeiss, Oberkochen, Germany; CCD camera, Hamamatsu, Tokyo, Japan; Infrapatch set-up, Luigs & Neumann, Ratingen, Germany; two HEKA EPC 9 Double patch-clamp ampli- fiers, Lambrecht, Pfalz, Germany). Signals were filtered at 5 kHz, digitized at 10 kHz, and analyzed with PULSE soſtware (HEKA, Lambrech/Pfalz, Germany). We identified the recorded cells by their membrane and firing


Vol. 54 | No. 6 | 2013 Table 1. Average copy number (dPCR) or CT rps18 gabrd mir-132


qRT-PCR* rps18


gabrd mir-132 *CT


67.67 (20.26)


33.60 (0.89)


25.80 (2.17)


20.80 (1.71)


23.32 (3.64)


23.69 (2.32)


30.4 59.43 (24.54)


2.6


7.60 (4.28)


8.4 23.00 (2.94)


327.2 20.69 (1.85)


1246 27.15 (4.34)


499.3 24.85 (0.91)


value (qRT-PCR) for genes from different neuron types


dPCR NGFC CV% FSBC CV% PC CV% No template CV% 34.9


41.3 60.25 (21.04)


56.3


2.00 (1.22)


12.8 25.60 (3.91)


360.5 21.20 (2.32)


61.0 15.3


0.63 (1.06)


0.43 (0.53)


1.67 (0.58)


n 168.2 7 123.3 5 34.7


NGFC CV% FSBC CV% PC CV% No template CV% 499.3 baseline


- 2025 baseline


187.9 22.87 (2.35)


values after 15 pre-amplification cycles; STDEV in brackets.


characteristics (18) and also with post hoc light microscopic assessment.


Single cell harvesting At the end of recording, as much of the intra- cellular content as possible was aspirated into the recording pipette by application of a gentle negative pressure while maintaining the tight seal. Te pipette was then delicately removed to allow outside-out patch formation (19). Next, the content of the pipette (2 µl) was expelled into a low-adsorbtion test tube (Axygen, Tewksbury, MA). Te sample was snap-frozen in liquid nitrogen and stored or immediately used for reverse transcription (RT).


First-strand cDNA synthesis of mRNA RT was carried out in two steps. Te first step was done for 5 min at 65°C in a total reaction volume of 5 µL, containing 2 µL intracellular solution with the cytoplasmic contents of the neuron, 0.3 µL reverse primer (Bioneer Corporation, Daedong, Korea), 0.3 µL 10 mM dNTPs (Life Technologies, Foster City, CA), 1 µL 5X first-strand buffer, 0.3 µL 0.1 mol/L DTT, 0.3 µL RNase inhibitor (Life Technologies) and 100 U of reverse transcriptase (Superscript III, Invitrogen, Carlsbad, CA). RT primers were designed using the CLC Main Workbench (CLC Bio, Aarhus, Denmark) soſtware. RT primer sequences were:


rps18 5′-ATTAACAGCAAAGGCCCA-3′


gabrd 5′-CCCCTACTCTCTTTCTCT-3′


hspb1 5′-CAGGGAAGAGGACACCAA-3′


hmox1 5′-GAGAGACAAAGGAAGACA-3′


Te second step of the reaction was carried out at 50°C for 1 h and then the reaction was stopped by heating at 70°C for 15 min. Te RT reaction mix was stored at -20°C until PCR amplification.


328


First-strand cDNA synthesis of miRNA RT was performed using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies). Te miRNA from each individual cell was reverse transcribed in the presence of TaqMan MicroRNA Assays (Life Technologies). Seven µl reaction mixtures contained 0.2 µl dNTPs, 1 µl MultiScribe Reverse Transcriptase (50 U/µL), 1.5 µl 10X RT Buffer, 0.2 µl RNase Inhibitor (20 U/ µL), 1.5 µl 5X RT primer, 0.6 µl nuclease- free water, and the template, for a total volume of 2 µl. RT was carried out with the following cycling parameters in a thermo- cycler (MyGenie; Bioneer): 16°C for 2 min, 42°C for 1 min, 50°C for 1 s, 45 cycles, then the samples were held at 85°C for 5 min.


Single cell qRT-PCR Traditional single cell qRT-PCR was carried out after preamplification of cDNA in a total volume of 20 µl (5 µL RT product, 1 µL TaqMan primer (rps18: Rn01428913_gH; gabrd: Rn01517017_g1; hsa-miR-132: 000457), 10 µL TaqMan PreAmp Master Mix (Life Technologies) and 4.5 µL nuclease-free water) in a MyGenie 32 Termal Block (Bioneer) with the following cycling protocol: 10 min hot start at 95°C, 2 min at 55°C, 2 min at 72°C followed by 15 cycles (15 s at 95°C, 4 min at 60°C), and finally 10 min at 99°C for inactivation of the enzyme. Aſter preamplification, qRT-PCR was carried out in an Exicycler 96 (Bioneer) in a total volume of 20 µL containing 1.5 µL preamplified PCR mixture, 1 µL TaqMan primer and 10 µL FastStart TaqMan Probe Master (Roche Diagnostics GmbH, Mannheim, Germany). Te following protocol was used: 3 min at 95°C followed by 45 cycles (40 s at 95°C, 40 s at 56°C, and 1 min at 72°C). Te Exicycler 96 Analysis Soſtware (Bioneer) determined a cycle threshold (CT


) value, which identified the first


cycle at which the fluorescence was detected above the baseline for that sample. For each gene, average threshold cycle (CT


) values were presented (Table 1). www.BioTechniques.com - baseline 509.8 baseline - - 5 n 6 2 4


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