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 (A75-05-24).
1. Preliminary Considerations
Note: To avoid contaminations, conform to the following recommendations before undertaking single cell RT-PCR after patch-clamp.
2. Primer Design
Note: Multiplex RT-PCR relies on two amplification steps. During the first PCR, all the genes of interest are co-amplified by mixing together all PCR primers. To detect reliably transcripts from single cells, it is essential to design efficient and selective PCR primers. The use of nested (internal) primers for the second rounds of PCR improves both the specificity and the efficiency of the amplification.
3. Preparation of RT Reagents
4. PCR Validation
5. Preparation and Validation of Intracellular Patch-clamp Solution
Note: The following protocol describes the preparation and validation of a K+/gluconate based internal solution, but virtually any type of patch-clamp solution can be used as long as it does not hamper the efficiency of the RT-PCR. Wearing gloves is mandatory to obtain an RNase-free internal solution.
6. Acute Slice Preparation
Note: This protocol describes the slicing procedure for juvenile (i.e., less than 28 postnatal days) male and female mice. Other cutting solutions, such as sucrose-based artificial cerebrospinal fluid (aCSF) are also usable40.
7. Single Cell RT-PCR after Patch-clamp Recording
8. Histochemical Staining of the Recorded Cell (Optional)
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 interneurons3,5,11. The cyclooxygenase-2 enzyme (COX-2) was used as a marker of the pyramidal cell sub-population3,16.
The ATP1α1-3 subunits of the Na+/K+ ATPase and the Kir6.2 and SUR1 subunits of the ATP-sensitive K+ channels were chosen to evaluate the relationship between neuronal activity and metabolic states43. Since Kir6.2 is an intron-less gene, the SOM intron was included as a genomic control. The protocol has been tested on 1 ng of reverse transcribed total RNA extracted from mouse whole brain, which corresponds approximately to the transcripts of 20 cells44. The multiplex PCR produced 12 amplicons of the expected size (Figure 3 and Table 1) demonstrating the sensitivity and efficiency of the protocol. The negative control performed without RNA produced no amplicon (data not shown).
A layer V pyramidal neuron was visually identified by its large soma and a prominent apical dendrite (Figure 5A, inset). Whole cell recording revealed typical electrophysiological properties of layer V regular spiking neurons5,45,46 with, notably, a low input resistance, long-lasting action potentials, and pronounced spike frequency adaptation (Figure 5A). The molecular analysis of this neuron revealed the expression of vGluT1 (Figure 5B), confirming its glutamatergic phenotype42,46. This neuron also expressed SOM, ATP1α1, and 3 subunits of the Na+/K+ ATPase. Biocytin labeling of the recorded neurons confirmed a pyramidal morphology (Figure 5D).
As expected from glutamatergic neurons, the molecular analysis of 26 layer V pyramidal cells revealed expression of VGluT1, but neither of the two GADs (Figure 5C). Markers of interneurons were rarely observed5,42,46. COX-2, chiefly expressed by layer II-III pyramidal cells3,16, was not detected in layer V pyramidal cells. The ATP1α1 and 3 subunits were more frequently observed than the ATP1α2 subunit3. The Kir6.2 and SUR1 subunits were rarely detected in layer V pyramidal neurons, which is consistent with their preferential expression in upper layers3,43. Care was taken not to harvest the nuclei, resulting in a rare detection of SOM introns (3 out of 26 cells, i.e., 12%).
Figure 1: Dedicated box for single cell RT-PCR. List of materials reserved for RT-PCR after patch-clamp. (1) Borosilicate glass capillaries, (2) 20 µL long, fine, flexible tips, (3) 20 µL micropipette, (4) home-made expeller, (5) 500 µL PCR tubes, (6) 10 µL aerosol resistant filter tips, and (7) permanent markers. Please click here to view a larger version of this figure.
Figure 2: PCR primer design strategy. (A) Schematic representation of the coding sequence of COX 2 cDNA aligned to the mouse chromosome 2 sequence containing the COX 2 gene. Exons are represented by black boxes and introns on gDNA by white boxes. Note the gaps in the cDNA corresponding to the intronic sequences on gDNA. Representative examples of bad and good oligonucleotides represented as red and green arrows, respectively. Right and left arrows denote forward and reverse primers. (B) Sequences and secondary structures of the oligonucleotides shown in (A). Left and right panels indicate hairpin self-complementarity and self-dimer formation, respectively. The B1 oligonucleotide displays both a strong hairpin self-complementarity and dimer formation whereas the B2 oligonucleotide only shows dimer formation. Both B3 and B4 oligonucleotides have no hairpin self-complementarity and no dimer formation; they are intron overspanning (A) and have been selected as potential PCR primers (see Table 1). Please click here to view a larger version of this figure.
Figure 3: Validation of multiplex PCR. 1 ng of total RNA from mouse whole brain was subjected to a reverse transcription followed by two rounds of PCR amplification. The 12 PCR products were resolved in separate lanes by agarose gel electrophoresis in parallel with Φx174 digested by HaeIII. The second PCR products had sizes (in bp) predicted by the sequences: 153 (vGluT1), 248 (GAD65), 255 (GAD67), 181 (COX 2), 220 (NPY), 146 (SOM), 183 (ATP1a1), 213 (ATP1a2), 128 (ATP1a3), 342 (Kir6.2), 211 (SUR1), and 182 (SOMint). Please click here to view a larger version of this figure.
Figure 4: Patch-clamp harvesting in brain slices. Once a cell is visually identified, the recording pipette is approached with a positive pressure in order to avoid cellular debris contamination at the tip of the pipette. Note the dimple on the membrane of this neuron. Positive pressure is then interrupted in order to form a cell-attached configuration with a Gigaohm tight seal. Brief suctions are applied to switch to whole-cell configuration. At the end of the recording, the cytoplasm is harvested by applying a gentle negative pressure into the pipette while maintaining the tight seal. Note the shrinkage of the cell body during the harvesting procedure. The recording pipette is then gently withdrawn to form an outside-out patch, which favors cell membrane closure for subsequent biocytin revelation, and preservation of harvested material in the patch pipette. Reproduced from Cauli and Lambolez47 with permission from the Royal Society of Chemistry. Please click here to view a larger version of this figure.
Figure 5: Characterization of layer V neocortical pyramidal cells. (A) Membrane potential response of a layer V pyramidal cell induced by current pulses of -100, -40, -10, +60, +120, and +500 pA (bottom traces). The neuron displays a weak potential deflection after the initial hyperpolarizing response (upper traces). In response to a supraliminal depolarization, the neuron discharged long-lasting action potentials with slowly developing after-hyperpolarization (upper trace). Near saturation, the neuron discharged at a low frequency and exhibited a pronounced adaptation (upper gray trace). Inset: infrared pictures of the recorded layer V pyramidal neuron. The pial surface is upward. Scale bar: 20 µm. (B) Multiplex RT-PCR analysis revealing expression of vGluT1, SOM, ATP1α1, and ATP1α3. (C) Gene expression profile in a sample of 26 layer V pyramidal cells. vGluT1 was expressed in all neurons while GAD65, GAD67, and COX2 were never detected. NPY, SOM, Kir6.2, SUR1, and SOMint were rarely observed. Pyramidal cells expressed ATP1α3, 1 subunit of the Na+/K+ ATPase, and ATP1α2 to a lesser extent. (D) Maximum intensity projection of confocal images showing biocytin labeling of the neuron recorded in (A). Please click here to view a larger version of this figure.
Gene / GenBank number | External primers | Size (bp) | Internal primers | Size (bp) | ||
vGlut1 NM_182993 |
Sense, -113 | 259 | Sense, -54 | 153 | ||
GGCTCCTTTTTCTGGGGCTAC | ATTCGCAGCCAACAGGGTCT | |||||
Anti-sense, 126 | Anti-sense, 79 | |||||
CCAGCCGACTCCGTTCTAAG | TGGCAAGCAGGGTATGTGAC | |||||
GAD65 NM_008078 |
Sense, 99 | 375 | Sense, 219 | 248 | ||
CCAAAAGTTCACGGGCGG | CACCTGCGACCAAAAACCCT | |||||
Anti-sense, 454 | Anti-sense, 447 | |||||
TCCTCCAGATTTTGCGGTTG | GATTTTGCGGTTGGTCTGCC | |||||
GAD67 NM_008077 |
Sense, 529 | 598 | Sense, 801 | 255 | ||
TACGGGGTTCGCACAGGTC | CCCAGAAGTGAAGACAAAAGGC | |||||
Anti-sense, 1109 | Anti-sense, 1034 | |||||
CCCAGGCAGCATCCACAT | AATGCTCCGTAAACAGTCGTGC | |||||
COX 2 NM_011198 |
Sense, 199 | 268 | Sense, 265 | 181 | ||
CTGAAGCCCACCCCAAACAC | AACAACATCCCCTTCCTGCG | |||||
Anti-sense, 445 | Anti-sense, 426 | |||||
CCTTATTTCCCTTCACACCCAT | TGGGAGTTGGGCAGTCATCT | |||||
NPY NM_023456 |
Sense, 16 | 294 | Sense, 38 | 220 | ||
CGAATGGGGCTGTGTGGA | CCCTCGCTCTATCTCTGCTCGT | |||||
Anti-sense, 286 | Anti-sense, 236 | |||||
AAGTTTCATTTCCCATCACCACAT | GCGTTTTCTGTGCTTTCCTTCA | |||||
SOM NM_009215 |
Sense, 43 | 208 | Sense, 75 | 146 | ||
ATCGTCCTGGCTTTGGGC | GCCCTCGGACCCCAGACT | |||||
Anti-sense, 231 | Anti-sense, 203 | |||||
GCCTCATCTCGTCCTGCTCA | GCAAATCCTCGGGCTCCA | |||||
ATP1α1 NM_144900 |
Sense, 1287 | 288 | Sense, 1329 | 183 | ||
CAGGGCAGTGTTTCAGGCTAA | TAAGCGGGCAGTAGCGGG | |||||
Anti-sense, 1556 | Anti-sense, 1492 | |||||
CCGTGGAGAAGGATGGAGC | AGGTGTTTGGGCTCAGATGC | |||||
ATP1α2 NM_178405 |
Sense, 1392 | 268 | Sense, 1430 | 213 | ||
AGTGAGGAAGATGAGGGACAGG | AAATCCCCTTCAACTCCACCA | |||||
Anti-sense, 1640 | Anti-sense, 1623 | |||||
ACAGAAGCCCAGCACTCGTT | GTTCCCCAAGTCCTCCCAGC | |||||
ATP1α3 NM_144921 |
Sense, 127 | 216 | Sense, 158 | 128 | ||
CGGAAATACAATACTGACTGCGTG | TGACACACAGTAAAGCCCAGGA | |||||
Anti-sense, 324 | Anti-sense, 264 | |||||
GTCATCCTCCGTCCCTGCC | CCACAGCAGGATAGAGAAGCCA | |||||
Kir6.2 NM_010602 |
Sense, 306 | 431 | Sense, 339 | 342 | ||
CGGAGAGGGCACCAATGT | CATCCACTCCTTTTCATCTGCC | |||||
Anti-sense, 719 | Anti-sense, 663 | |||||
CACCCACGCCATTCTCCA | TCGGGGCTGGTGGTCTTG | |||||
SUR1 NM_011510 |
Sense, 1867 | 385 | Sense, 2041 | 211 | ||
CAGTGTGCCCCCCGAGAG | ATCATCGGAGGCTTCTTCACC | |||||
Anti-sense, 2231 | Anti-sense, 2231 | |||||
GGTCTTCTCCCTCGCTGTCTG | GGTCTTCTCCCTCGCTGTCTG | |||||
SOMint X51468 |
Sense, 8 | 240 | Sense, 16 | 182 | ||
CTGTCCCCCTTACGAATCCC | CTTACGAATCCCCCAGCCTT | |||||
Anti-sense, 228 | Anti-sense, 178 | |||||
CCAGCACCAGGGATAGAGCC | TTGAAAGCCAGGGAGGAACT |
Table 1. Sequences of first and second PCR primers. The position of each PCR primer and their sequences are given from 5' to 3'. Except for the somatostatin intron, position 1 corresponds to the first base of the start codon of each gene.
MACAW v.2.0.5 | NCBI | Multiple alignement for primer design | |
Dithiothreitol | VWR | 443852A | RT |
Random primers | Sigma-Aldrich (Merck) | 11034731001 | RT |
dNTPs | GE Healthcare Life Sciences | 28-4065-52 | RT and PCR |
RNasin Ribonuclease Inhibitors | Promega | N2511 | RT |
SuperScript II Reverse Transcriptase | Invitrogen | 18064014 | RT |
Taq DNA Polymerase | Qiagen | 201205 | PCR |
Mineral Oil | Sigma-Aldrich (Merck) | M5904-5ML | PCR |
PCR primers | Sigma-Aldrich (Merck) | PCR / desalted and diluted at 200 µM | |
Tubes, 0.5 mL, flat cap | ThermoFisher Scientific | AB0350 | RT and PCR |
BT10 Series – 10 µL Filter Tip | Neptune Scientific | BT10 | RT and PCR |
BT20 Series – 20 µL Filter Tip | Neptune Scientific | BT20 | RT and PCR |
BT200 Series – 200 µL Filter Tip | Neptune Scientific | BT200 | RT and PCR |
BT1000 Series – 1000 µL Filter Tip | Neptune Scientific | BT1000.96 | RT and PCR |
DNA Thermal Cylcer | Perkin Elmer Cetus | PCR | |
Ethidium Bromide | Sigma-Aldrich (Merck) | E1510-10ML | Agarose gel electrophoresis |
Tris-Borate-EDTA buffer | Sigma-Aldrich (Merck) | T4415-1L | Agarose gel electrophoresis |
UltraPure Agarose | Life Technologies | 16500-500 | Agarose gel electrophoresis |
ΦX174 DNA-Hae III Digest | NEB (New England BioLabs) | N3026S | Agarose gel electrophoresis |
EDA 290 | Kodak | Agarose gel electrophoresis | |
Electrophoresis Power supply EPS 3500 | Pharmacia Biotech | Agarose gel electrophoresis | |
Midi Horizontal Elecrophoresis Unit Model SHU13 | Sigma-Aldrich (Merck) | Agarose gel electrophoresis | |
Smooth paper with satin appearance | Fisherbrand | 1748B | Patch clamp internal solution |
Potassium Hydroxyde | Sigma-Aldrich (Merck) | 60377 | Patch clamp internal solution |
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid | Sigma-Aldrich (Merck) | E3889 | Patch clamp internal solution |
HEPES | Sigma-Aldrich (Merck) | H4034 | Patch clamp internal solution |
Potassium D-gluconate | Sigma-Aldrich (Merck) | G4500 | Patch clamp internal solution |
Magnesium chloride solution | Sigma-Aldrich (Merck) | M1028 | Patch clamp internal solution |
5500 Vapor Pressure Osmometer | Wescor | Patch clamp internal solution | |
Biocytin | Sigma-Aldrich (Merck) | B4261 | Patch clamp internal solution |
Sucrose | Sigma-Aldrich (Merck) | S5016 | Slice preparation |
D-(+)-Glucose monohydrate | Sigma-Aldrich (Merck) | 49159 | Slice preparation |
Sodium chloride | Sigma-Aldrich (Merck) | S6191 | Slice preparation |
Potassium chloride | Sigma-Aldrich (Merck) | 60128 | Slice preparation |
Sodium bicarbonate | Sigma-Aldrich (Merck) | 31437-M | Slice preparation |
Sodium phosphate monobasic | Sigma-Aldrich (Merck) | S5011 | Slice preparation |
Magnesium chloride solution | Sigma-Aldrich (Merck) | 63069 | Slice preparation |
Calcium chloride solution | Sigma-Aldrich (Merck) | 21115 | Slice preparation |
Kynurenic acid | Sigma-Aldrich (Merck) | K3375 | Slice preparation |
Isoflurane | Piramal Healthcare UK | Slice preparation | |
VT 1000S | Leica Biosystems | 14047235613 | Slice preparation |
Hydrogen peroxide solution | Sigma-Aldrich (Merck) | H1009 | Patch Clamp set-up cleaning |
Thin Wall Glass Capillaries with filament | World Precision Instruments | TW150F-4 | Patch Clamp |
PP-83 | Narishige | Patch Clamp | |
Eppendorf Microloader | Eppendorf | 5242956003 | Patch Clamp |
BX51WI Upright microscope | Olympus | Patch Clamp | |
XC-ST70/CE CCD B/W VIDEO CAMERA | Sony | Patch Clamp | |
Axopatch 200B Amplifier | Molecular Devices | Patch Clamp | |
Digidata 1440 | Molecular Devices | Patch Clamp | |
pCLAMP 10 software suite | Molecular Devices | Patch Clamp | |
10 mL syringe | Terumo | SS-10ES | Expelling |
E Series with Straight Body (Holder) | Phymep | 64-0997 | Expelling |
Sodium phosphate dibasic | Sigma-Aldrich (Merck) | S7907 | Histochemical revelation |
Sodium phosphate monobasic | Sigma-Aldrich (Merck) | S8282 | Histochemical revelation |
Paraformaldehyde | Sigma-Aldrich (Merck) | P6148 | Histochemical revelation |
Triton X-100 | Sigma-Aldrich (Merck) | X100 | Histochemical revelation |
Gelatin from cold water fish skin | Sigma-Aldrich (Merck) | G7041 | Histochemical revelation |
Streptavidin, Alexa Fluor 488 conjugate | ThermoFisher Scientific | S11223 | Histochemical revelation |
24-well plate | Greiner Bio-One | 662160 | Histochemical revelation |
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.
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.
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.