Name: single cell multiplex reverse transcription polymerase chain reaction after patch-clamp
Uploaded: 2018-06-20T13:00:00
Description: Watch this Scientific Journal Video about Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp at JoVE.com

We thank Dr. Alexandre Mourot for his comments on the manuscript. This work was supported by grants from the Agence Nationale de la Recherche (ANR 2011 MALZ 003 01; ANR-15-CE16-0010 and ANR-17-CE37-0010-03), BLG is supported by fellowship from the Fondation pour la Recherche sur Alzheimer. We thank the animal facility of the IBPS (Paris, France).

All experimental procedures using animals were performed in strict accordance with French regulations (Code Rural R214/87 to R214/130) and conformed to the ethical guidelines of both the European Economic Community (86/609/EEC) and the French National Charter on the ethics of animal experimentation. All protocols were approved by the Charles Darwin ethics committee and submitted to the French Ministry of Education and Research (Approval 2015 061011367540). The IBPS animal facility is accredited by the French authorities ...

Single cell multiplex RT-PCR after patch-clamp can simultaneously and reliably probe the expression of more than 30 genes in electrophysiologically identified cells5. Analyzing gene expression at the single cell level requires highly efficient PCR primers. One of the most limiting steps is collection of the cell&#39;s content. Its efficiency depends on the diameter of the patch pipette tip, which must be as large as possible while matching the cell size. Pipettes with a 1-2 &#181;m open tip diamet...

The cerebral cortex comprises numerous cell types involved in various physiological processes. Their identification and characterization, a prerequisite to the understanding of their specific functions, can be very challenging given the large morphological, physiological, and molecular diversity that characterizes cortical cell types1,2,3,4.
Single-cell multiplex RT-PCR is based on the combination of patch-clamp and RT-PCR techniques. It can probe simultaneously the expression of more than 30 predefined genes in electrophysiologically identified cells5. The inclusion of a neuronal tracer in the recording pipette further allows the morphological characterization of the recorded cells after histochemical revelation6,7,8,9,10. It is a very useful technique for the classification of neuronal types based on multivariate analysis of their phenotypic traits5,9,10,11,12,13,14. Single cell multiplex RT-PCR is also suited to the characterization of non-neuronal cells such as astrocytes15,16,17, and can be virtually applied to every brain structure18,19,20,21,22,23 and cell type, assuming they can be recorded in whole-cell configuration.
This technique is very convenient for the identification of cellular sources and/or targets of transmission systems7,8,15,16,20,21,24,25,26,27,28, especially when specific antibodies are lacking. It relies on patch-clamp recordings from visually identified cells29, and thus also allows the targeting of cells in a specific cellular environment8,15,16. Furthermore since the cytoarchitecture of brain tissue is preserved in brain slices, this approach also enables study of the anatomical relationships of the characterized cells with neuronal and non-neuronal elements7,8,18.
Since this technique is limited by the amount of harvested cytoplasm and by the efficiency of the RT, the detection of mRNA expressed at low copy number can be difficult. Although other approaches based on RNaseq technology allow to analyze the whole transcriptome of single cells3,4,30,31, they need high-throughput expensive sequencers not necessarily available to every laboratory. Since the single cell multiplex RT-PCR technique uses end-point PCR, it only requires widely available thermocyclers. It can be easily developed in laboratories equipped with electrophysiological set-ups and does not require expensive equipment. It can provide, within one day, a qualitative analysis of the expression of a predefined set of genes. Thus, this approach offers an easy access to the molecular characterization of single cells in a rapid manner.

<table>
	<tbody>
		<tr>
			<td>MACAW v.2.0.5</td>
			<td>NCBI</td>
			<td></td>
			<td>Multiple alignement for primer design</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>VWR</td>
			<td>443852A</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Random primers</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>11034731001</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-4065-52</td>
			<td>RT and PCR</td>
		</tr>
		<tr>
			<td>RNasin Ribonuclease Inhibitors</td>
			<td>Promega</td>
			<td>N2511</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064014</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>Qiagen</td>
			<td>201205</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>M5904-5ML</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td></td>
			<td>PCR / desalted and diluted at 200 &#181;M</td>
		</tr>
		<tr>
			<td>Tubes, 0.5 mL, flat cap</td>
			<td>ThermoFisher Scientific</td>
			<td>AB0350</td>
			<td>RT and PCR</td>
		</tr>
		<tr>
			<td>BT10 Series - 10 &#181;L Filter Tip</td>
			<td>Neptune Scientific</td>
			<td>BT10</td>
			<td>RT and PCR</td>
		</tr>
		<tr>
			<td>BT20 Series - 20 &#181;L Filter Tip</td>
			<td>Neptune Scientific</td>
			<td>BT20</td>
			<td>RT and PCR</td>
		</tr>
		<tr>
			<td>BT200 Series - 200 &#181;L Filter Tip</td>
			<td>Neptune Scientific</td>
			<td>BT200</td>
			<td>RT and PCR</td>
		</tr>
		<tr>
			<td>BT1000 Series - 1000 &#181;L Filter Tip</td>
			<td>Neptune Scientific</td>
			<td>BT1000.96</td>
			<td>RT and PCR</td>
		</tr>
		<tr>
			<td>DNA Thermal Cylcer</td>
			<td>Perkin Elmer Cetus</td>
			<td></td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>E1510-10ML</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Tris-Borate-EDTA buffer</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>T4415-1L</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Life Technologies</td>
			<td>16500-500</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>&#934;X174 DNA-Hae III Digest</td>
			<td>NEB (New England BioLabs)</td>
			<td>N3026S</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>EDA 290</td>
			<td>Kodak</td>
			<td></td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Electrophoresis Power supply EPS 3500</td>
			<td>Pharmacia Biotech</td>
			<td></td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Midi Horizontal Elecrophoresis Unit Model SHU13</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td></td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Smooth paper with satin appearance</td>
			<td>Fisherbrand</td>
			<td>1748B</td>
			<td>Patch clamp internal solution</td>
		</tr>
		<tr>
			<td>Potassium Hydroxyde</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>60377</td>
			<td>Patch clamp internal solution</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>E3889</td>
			<td>Patch clamp internal solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>H4034</td>
			<td>Patch clamp internal solution</td>
		</tr>
		<tr>
			<td>Potassium D-gluconate</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>G4500</td>
			<td>Patch clamp internal solution</td>
		</tr>
		<tr>
			<td>Magnesium chloride solution</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>M1028</td>
			<td>Patch clamp internal solution</td>
		</tr>
		<tr>
			<td>5500 Vapor Pressure Osmometer</td>
			<td>Wescor</td>
			<td></td>
			<td>Patch clamp internal solution</td>
		</tr>
		<tr>
			<td>Biocytin</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>B4261</td>
			<td>Patch clamp internal solution</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>S5016</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose monohydrate</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>49159</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>S6191</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>60128</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>31437-M</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>S5011</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>Magnesium chloride solution</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>63069</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>Calcium chloride solution</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>21115</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>Kynurenic acid</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>K3375</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare UK</td>
			<td></td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>VT 1000S</td>
			<td>Leica Biosystems</td>
			<td>14047235613</td>
			<td>Slice preparation</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>H1009</td>
			<td>Patch Clamp set-up cleaning</td>
		</tr>
		<tr>
			<td>Thin Wall Glass Capillaries with filament</td>
			<td>World Precision Instruments</td>
			<td>TW150F-4</td>
			<td>Patch Clamp</td>
		</tr>
		<tr>
			<td>PP-83</td>
			<td>Narishige</td>
			<td></td>
			<td>Patch Clamp</td>
		</tr>
		<tr>
			<td>Eppendorf Microloader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>Patch Clamp</td>
		</tr>
		<tr>
			<td>BX51WI Upright microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Patch Clamp</td>
		</tr>
		<tr>
			<td>XC-ST70/CE CCD B/W VIDEO CAMERA</td>
			<td>Sony</td>
			<td></td>
			<td>Patch Clamp</td>
		</tr>
		<tr>
			<td>Axopatch 200B Amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Patch Clamp</td>
		</tr>
		<tr>
			<td>Digidata 1440</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Patch Clamp</td>
		</tr>
		<tr>
			<td>pCLAMP 10 software suite</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Patch Clamp</td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>Terumo</td>
			<td>SS-10ES</td>
			<td>Expelling</td>
		</tr>
		<tr>
			<td>E Series with Straight Body (Holder)</td>
			<td>Phymep</td>
			<td>64-0997</td>
			<td>Expelling</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>S7907</td>
			<td>Histochemical revelation</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>S8282</td>
			<td>Histochemical revelation</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>P6148</td>
			<td>Histochemical revelation</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>X100</td>
			<td>Histochemical revelation</td>
		</tr>
		<tr>
			<td>Gelatin from cold water fish skin</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>G7041</td>
			<td>Histochemical revelation</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 488 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>S11223</td>
			<td>Histochemical revelation</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662160</td>
			<td>Histochemical revelation</td>
		</tr>
	</tbody>
</table>

single cell multiplex reverse transcription polymerase chain reaction after patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

A representative validation of multiplex RT-PCR is shown in Figure 3. The protocol was designed to probe simultaneously the expression of 12 different genes. The vesicular glutamate transporter vGluT1 was taken as a positive control for glutamatergic neurons42. The GABA synthesizing enzymes (GAD65 and GAD 67), Neuropeptide Y (NPY), and Somatostatin (SOM) were used as markers of GABAergic interneurons3,</sup...

Watch this Scientific Journal Video about Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp at JoVE.com

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

This method can help answer key questions in neuroscience research, such as characterizing neuro population or identifying sources and targets of transmission systems. The main advantage of this technique is that it allows to determine the expression profile of a pre-selected set of genes from a single cell quickly and easily. So this method can provide insight into the random diversity mouse.It can also be applied to other cell types, such as astrocytes and in many animal models. This demonstration of this method is critical as the arresting and aspiring steps can be difficult to learn because the data needs a quantity of cytoplasm to be analyzed. Before sectioning, prepare a dissection kit containing surgical scissors, fine iris scissors, two spatulas, forceps, a disc of paper filter, and cyanoacrylate glue.Then saturate the ice cold cutting solution with carbogyn and cool down the cutting chamber at negative 20 degrees Celsius. Next, remove the animal's scalp and open the skull. Extract the brain carefully and place it into a small beaker filled with carbogenated ice cold cutting solution.Afterward, remove the cutting chamber from negative 20 degrees Celsius and remove the moisture with a paper towel. Carefully dissect the brain to isolate the region of interest. Glue it on to the cutting chamber and add the carboginated, ice cold cutting solution.Using a vibratone, section 300 micrometer thick slices. Transfer the slices to a resting chamber filled with oxygenated cutting solution at room temperature and allow them to recover for at least 30 minutes. For single cell RT-PCR and the targeted cell characterization, limit whole cell recording to no longer than 20 minutes in order to preserve the mRNAse.At the end of the recording prepare a 500 microliter PCR tube filled with two microliters of 5xRT mix and 0.5 microliters of 20xDTT. Spin it and store it on ice. Then harvest the cell's cytoplasm by applying a gentle negative pressure.Monitor and control the cell's content from getting into the pipette while maintaining a tight seal and avoid collecting the nucleus if some genes are considered. In the case of gene, it is essential to avoid the collection of the nucleus and to include a set of primers aimed at amplifying gDNA to prep for general contamination. If the nucleus is getting close to the tip of the pipette, release the negative pressure and move the pipette away.Ensure that the tight seal is preserved and restart the collection of the cytoplasm at the new pipette location. Next, withdraw the pipette gently to form an outside out patch to limit contamination by the extracellular debris and to favor the closure of the cell membrane for subsequent histo-chemical analysis. Keep in mind that nubs, micro-manipulators, computer keyboard or computer mouse, are proton charged sources of rnase contamination.If they have been touched during the recording, change the gloves. Subsequently, attach the pipette to the expeller, and expel its content into the PCR tube by applying positive pressure. Break the tip of the pipette into the PCR tube to help the collection of its content.Then, briefly centrifuge the tube add 0.5 microliters of RNAse inhibitor and 0.5 microlitres of RTAse. Mix gently, centrifuge again, and incubate overnight at 37 degrees Celsius. The next day, spin the tube, and store it at negative 80 degrees Celsius for up to several months.Until PCR analysis and performing the first amplication step. To validate PCR, mix and dilute a primer pair, add one micromolar with the pipette dedicated to the single cell RTPCR. Set the thermo cycler for three minutes at 95 degrees Celsius, run 40 cycles followed by a final elongation step at 72 degrees Celsius for five minutes.To minimize the formation of primer dimers, perform a hot start by placing the PCR tubes in the thermo cycler preheated at 95 degrees Celsius. After 30 seconds, quickly expel 20 microliters of the primer mix on top of the oil. Once the primer pair has been validated, test it with the multiplex protocol.Mix and dilute the external primer together at one micromolar. Store the multiplex primer mix at negative 20 degrees Celsius for up to several weeks. Next, prepare a pre-mix and place it on ice.Flick the PCR tube and spin it and add two drops of mineral oil. After three minutes at 95 degrees Celsius, run 20 PCR cycles. Perform a hot start as described with 20 microlitres of multiplex primer mix.Then, prepare a number of PCR tubes identical to the number of genes being analyzed. Prepare a pre mix using one microliter of the first PCR product per gene as template. Adjust all the volumes according to the number of genes to analyze and consider using 10%of extra volume to compensate for pipetting errors.Shake gently, spin the tube and dispatch 80 microliters of premix into each PCR tube. Add two drops of mineral oil per tube without touching the wall or the cap of the tubes. Run 35 PCR cycles then perform a hot start by expelling 20 microliters of internal primer mix.Analyze the second PCR projects by aragose gel electrophoresis. A layer five pyramidal neuron was visually identified by it's large soma and a prominent apical dendrite. Pull-cell recording revealed typical electro-physiological properties of layer five regular spiking neurons with a low input resistance, long lasting action potentials, and pronounced spike-frequency adaptation.The molecular analysis of this neuron revealed the expression of vGluT1, confirming its glutamitergic phenotype. This neuron also expressed somatostatin and the ATP1-alpha-1 and three sub-units of the sodium-potassium ATPAs. Biocytin labeling of the recorded neurons, confirmed a pyramidal morphology.As expected from the glutamitergic neurons, the molecular analysis of 26 layer-five pyramidal cells, revealed the expression of vGluT1 but neither of the two GADs. Once technique can be done within two days, if it's preformed properly. While attempting this procedure, it's important to remember to be very careful to avoid contaminations.After watching this video, you should have a good understanding of how to probe the expression of 10s of genes simultaneously from single cells characterized by patch clumps according to brain slices, after cytoplasm harvesting, reverse transcription, and hot-start PCRs. Don't forget that working with Ethidium bromide can be extremely hazardous and precautions such as wearing gloves and lab coats should always be taken while performing this procedure.

Research

JoVE Journal

Neuroscience

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The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

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