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Method Article

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

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DOI:

10.3791/57627

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June 20th, 2018

 ,  ,  , 
1UPMC Univ Paris 06, INSERM, CNRS, Neuroscience Paris Seine - Institut de Biologie Paris Seine (NPS - IBPS), Sorbonne Université

* These authors contributed equally

In This Article

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

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.

  1. Do not use plasmids containing genes of interest in the laboratory as they are a potential source of contamination.
  2. Reserve a lab bench away from the gel electrophoresis room to prepare PCRs.
  3. Dedicate a set of pipettes and aerosol resistant filter tips to manipulate DNA and RNA at concentrations lower than 1 ng/µL. Never use these to analyze PCR products.
  4. Always use RNase- and DNase-free products and wear gloves.
  5. Use dedicated chemicals for preparing internal patch-clamp solutions. Never use a spatula but rather weighing paper to weigh powders.
  6. Use a dedicated pH electrode and pH standard solutions, as they can be a source of contamination (e.g., RNase).
  7. Store borosilicate glass capillaries; 20 µL long, fine and flexible tips; a 20 µL micropipette; a home-made expeller (patch-clamp pipette holder attached to a 10 mL syringe); 500 µL PCR tubes; 10 µL aerosol resistant filter tips; and thin permanent markers in a dedicated box (Figure 1).

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.

  1. Retrieve the curated mRNA sequences of the genes of interest in NCBI Reference Sequence Database32 as well as those of their related family (https://www.ncbi.nlm.nih.gov/refseq/).
    1. Enter the name of the gene(s) and the species of interest as a query and click on the retrieved relevant sequence.
    2. To restrict the analysis to the coding sequence of the gene(s), click on Send to (upper right corner), and select Coding sequences and FASTA Nucleotide as format. Then click on Create File and save the FASTA sequence using a ".nt" file extension.
  2. Use MACAW software33 (Multiple Alignment Construction & Analysis Workbench) or any other multiple alignment software to determine the regions of homology.
    1. Click on File | New Project... and select DNA as the sequence type. To import the gene sequences, select Sequence | Import Sequence... and load a ".nt" file. Repeat the procedure for all related sequences.
    2. Click and drag all sequences in the Schematic window to select the entire sequences.
    3. Search the regions of homology by selecting Alignment | Search for Blocks... (CTRL+S). Select Segment pair overlap in Search Method. Adjust the Pairwise score cutoff to a relatively high value (e.g., 1,000) and the Min. seqs. per block to the total number of sequences to align and click on Begin.
    4. In the appearing Search Result window, select the result(s) with the highest MP-score and click on Link. If no result is found, decrease the Pairwise score cutoff and/or the Min. seqs. per block.
    5. To facilitate the visualization of homologous regions, select Alignment | Shading | Mean score.
    6. Select unlinked parts of the sequences and repeat the steps 2.2.3–2.2.4 to potentially determine other regions of homology with a lower MP-score.
    7. To obtain the most specific primers, select the regions with the least sequence homology.
  3. Fetch the sequences of all splice variants and repeat steps 2.2.1–2.2.6. Select their common regions for an overall detection. Consider alternative cassettes for dedicated splice variants analyses20,34,35,36.
  4. Align the mRNA sequences on the animal model genome using BLAST37 genomes (Basic Local Alignment Search Tool) to determine the intron-exon structure of the genes (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
    1. Select the BLAST mouse genome by clicking on Mouse and upload the retrieved FASTA sequence in the Enter Query Sequence section. In the Program Selection, select Optimize for Highly similar sequences (megablast), then click on the BLAST button.
    2. In the Descriptions section, select the alignment with the highest Identity (up to 100%). Make sure that it corresponds to the gene of interest as indicated in features of the Alignments section.
    3. Sort the alignment by Query start position in the same section to obtain the exons succession of the coding sequence. Note the start and end positions of all hits which roughly correspond to the position of the introns on the query sequence (Figure 2A).
  5. Select two different exons for the sense and antisense primers to differentiate easily cDNA from genomic DNA (gDNA) amplification based on a size criterion (Figure 2).
    Note: The amplification of gDNA can occur at a low frequency when the nucleus is harvested with the cytoplasm. Thus, it is essential to design intron overspanning primer pairs (Figure 2A). In case of intron-less genes, the collection of the nucleus is problematic as it can lead to potentially confounding results. To address this issue, include a set of primers aimed at amplifying an intronic sequence to probe for the presence of gDNA25. If the genomic control is positive, the results for all intron-less genes must be discarded.
  6. To achieve an efficient amplification, select primers generating amplicons with a length ideally comprised between 200 and 400 base pairs (Figure 2).
  7. To amplify simultaneously various cDNAs during the multiplex PCR step, design primers with a length between 18 and 24 nucleotides and a melting temperature (Tm) between 55 and 60 °C.
  8. To minimize the formation of secondary structures, which reduce the amplification yield, select sense and anti-sense primers with minimal hairpin and duplex lengths (Figure 2B).
  9. Design internal primers using the same criteria (steps 2.5–2.8).
  10. Verify the specificity of the PCR primers by aligning the primers on the Reference RNA Sequence Database of the organism of interest using a nucleotide BLAST.
    1. In BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), click on nucleotide BLAST. Paste the sequence of a primer in the Enter Query Sequence.
    2. In the Choose Search Set section, click on Others (nr etc.), select Reference RNA sequences (refseq_rna) in the Database option and specify the organism (e.g., Mus musculus (taxid:10090)) to restrict the analysis to the desired species.
    3. In Program Selection select Optimize for Somewhat similar sequences (blastn). In the Algorithm parameters section reduce the word size down to 7 to increase the chance to obtain hits.
  11. To design selective primers, keep only those whose sequence has at least 3 mismatches with undesired cDNAs when possible.
  12. Order desalted primers at a relatively high concentration (e.g., 200 µM) to ensure that all external primers can be mixed together at a final concentration of 1 µM.

3. Preparation of RT Reagents

  1. 5x RT-mix: Prepare in RNase-free water 400 µL of working RT mix solution (5x) containing random primers at 25 µM and dNTPs at 2.5 mM each. Store this working mix as 20 µL aliquots in 500 µL tubes.
  2. 20x Dithiothreitol (DTT): Prepare 1 mL of 0.2 M DTT in RNase-free water and store it as 50 µL aliquots.
  3. Store 2,500 U of RNase inhibitor (40 U/µL) and 10,000 U of reverse transcriptase (RTase, 200 U/µL) as 5 µL aliquots.
  4. To ensure an optimal quality of the RT reagents, store the aliquots at -80 °C. Use them for up to 10 cells and only for one day.

4. PCR Validation

  1. Prepare cDNAs by doing a reverse transcription on a relatively large amount of total RNA (typically 1 µg) extracted38 from the structure of interest.
    1. Do not use the pipettes dedicated to single cell RT-PCR for these preliminary steps but use the RNase-free pipettes. Dilute total RNA down to 1 µg/µL.
    2. In a 500 µL PCR tube, add 1 µL of diluted total RNA and 8 µL of RNase-free water. Denature at 95 °C for 1 min and cool down the tube on ice.
    3. Add 4 µL of RT 5x buffer supplied by the manufacturer, 4 µL of RT mix solution, 1 µL of RTase, 1 µL of RNase inhibitor, and 1 µL of 200 mM DTT (see Section 3).
    4. Flick the tube, spin it, and incubate overnight at 37 °C.
    5. Store the cDNAs at -80 °C for up to several years. Prepare an aliquot of cDNAs diluted to 1 ng/µL of total RNAs equivalents.
  2. Mix and dilute each primer pair at 1 µM with the pipettes dedicated to single cell RT-PCR. Use 20 µL of each primer mix for 100 µL PCRs.
  3. On ice, prepare for each primer pair a premix containing: water (q.s., 80 µL), 10 µL of 10X buffer, 1 µL of 100x dNTPs (50 µM each), 500–1,000 pg of cDNA diluted at 1 ng/µL, and 0.5 µL (2.5 U, 5 U/µL) of Taq polymerase.
  4. Use PCR tubes that tightly fit the wells of the PCR machine for an optimal temperature control. Add 2 drops (~100 µL) of mineral oil to the premix without touching the wall or the cap of the PCR tube.
  5. To minimize the formation of primer-dimers, perform a hot start by placing the PCR tubes in the thermocycler pre-heated at 95 °C. After 30 s, quickly expel 20 µL of the primer mix on top of the oil.
  6. After 3 min at 95 °C, run 40 cycles (95 °C, 30 s; 60 °C, 30 s; 72 °C, 35 s) followed by a final elongation step at 72 °C for 5 min.
  7. Analyze 10 µL of each PCR products by agarose gel electrophoresis (2%, weight/volume)39. If some PCR products do not have the expected size, re-design other primers (see Section 2).
    Caution: Ethidium bromide is an intercalating chemical that can induce DNA mutations. Consult the local safety office before using it. Always wear gloves when manipulating it. Use a commercially available solution of ethidium bromide (10 mg/mL) solution instead of powder to minimize the risk of inhalation. Similarly, UV light can be harmful, so make sure to wear UV protection safety glasses or a mask. Withdraw soiled gels, tips, gloves, and buffer in containers dedicated to ethidium waste.
  8. Once all primer pairs have been individually validated, test the multiplex protocol (Figure 3).
    1. Mix and dilute all external primers together at 1 µM. Store the multiplex primers mix at -20 °C for up to several weeks.
    2. On ice, prepare a premix containing: water (q.s., 80 µL), 10 µL of 10X buffer, 1 µL of 100x dNTPs (50 µM of each), 500–1,000 pg of cDNAs diluted at 1 ng/µL, and 0.5 µL (2.5 U, 5 U/µL) of Taq polymerase.
    3. Flick the PCR tube, spin it, and add two drops (~100 µL) of mineral oil.
    4. Perform a hot start as described in step 4.5 with 20 µL of multiplex primer mix. After 3 min at 95 °C, run 20 PCR cycles identical to those of step 4.6.
    5. Prepare a number of PCR tubes identical to the number of genes to analyze. Prepare a premix as in step 4.8.2, but using 1 µL 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.
    6. Shake gently, spin the tube, dispatch 80 µL of premix in each PCR tube, and add two drops (~100 µL) of mineral oil per tube without touching the wall or the cap of the tubes.
    7. Perform a hot start (see step 4.5) by expelling 20 µL of internal primer mix prepared in step 4.2. Run 35 PCR cycles identical to step 4.6.
    8. Analyze the second PCR products by agarose gel electrophoresis. Make sure that every second PCR generates an amplicon of the expected size. In case of inefficient or nonspecific amplifications, re-design new primers (steps 2.5–2.12) and validate them (steps 4.2–4.8).

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.

  1. For 60 mL of internal patch-clamp recording solution, prepare extemporaneously 10 mL of 0.1 M KOH.
  2. Dissolve 11.4 mg of EGTA (0.5 mM final) in 0.9 mL of 0.1 M KOH.
  3. Add 40 mL RNase-free water and dissolve 2.02 g of K-gluconate (144 mM final), 143 mg of HEPES (10 mM final), and 180 µL of 1 M MgCl2 (3 mM final).
  4. Adjust the pH to 7.2 by adding ~6 mL of 0.1 M KOH.
  5. Adjust the osmolarity to 295 mOsm with ~13 mL of RNase-free water.
  6. Since the internal solution is also used as a buffer for the reverse transcription, carefully control Mg2+ concentration. Compensate for a lower Mg2+ concentration in the internal solution by adding MgCl2 afterwards to reach a final concentration of 2 mM in the RT reaction.
  7. To label the harvested cell after patch-clamp recording, add 2–5 mg/mL of RNase-free biocytin to the internal solution described above (steps 5.1–5.5).
  8. Filter (0.22 µm pore size) the internal solution and store it at -80 °C as 100–250 µL aliquots.
  9. To validate the use of the internal solution for single-cell RT-PCR, add 0.5 µL of total RNAs diluted at 1 ng/µL to 6 µL of patch-clamp solution and leave it on the bench for about 30 min.
  10. With a 2 µL micropipette, add 2 µL of 5x RT-mix, 0.5 µL of 20x DDT, 0.5 µL RNase inhibitor, and 0.5 µL RTase, and incubate overnight at 37 °C.
  11. Spin the tube. On ice add water (q.s., 80 µL), 10 µL of 10X buffer, and 0.5 µL (2.5 U, 5 U/µL) of Taq polymerase.
  12. Perform 40 cycles of PCR using a set of validated primers (steps 4.4–4.6).
  13. Analyze the PCR products by agarose gel electrophoresis (see step 4.7) and verify that they have the expected size.
    Note: Omitting the 0.5 µL of total RNA and replacing it with 0.5 µL RNase-free water will ensure that the internal solution is free of RNA and/or DNA contaminations.

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.

  1. Prepare 2 L of aCSF containing (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 Glucose, and 15 Sucrose. To reduce glutamatergic activity during slice preparation, prepare a cutting solution by adding 1 mM of kynurenic acid.
  2. Before slicing, prepare a dissection kit containing surgical scissors, fine iris scissors, two spatulas, forceps, a disc of paper filter, and cyanoacrylate glue.
  3. Use ice-cold cutting solution saturated with O2/CO2 (95%/5%) and cool down the cutting chamber at -20 °C.
  4. Anesthetize the mouse with a small paper towel soaked with isoflurane. After ~2 min, make sure that the mouse is under deep anesthesia by verifying the absence of response to paw pinch.
  5. Quickly decapitate the mouse. Remove the scalp and open the skull. Extract the brain carefully and place it into a small beaker filled with ice cold (~4 °C) cutting solution oxygenated with O2/CO2.
  6. Remove the cutting chamber from -20 °C conditions and remove moisture with a paper towel.
  7. Carefully dissect the brain to isolate the region of interest, glue it on the cutting chamber, and add ice-cold cutting solution oxygenated with O2/CO2.
  8. Cut 300 µm thick slices using a vibratome. Transfer them at room temperature in a resting chamber filled with oxygenated cutting solution and allow them to recover for at least 0.5 h.

7. Single Cell RT-PCR after Patch-clamp Recording

  1. Clean the perfusion system regularly with ~100 mL of 30% H2O2 solution and rinse extensively with ~500 mL of distilled water to avoid bacteria growth, as it is an overlooked source of RNase contamination.
  2. Chlorinate the filament with a patch pipette filled with concentrated bleach every harvesting day. Do not use a micropipette to fill the patch pipette but rather a 20 µL long, fine, flexible tip attached to a 1 mL syringe. Then, rinse it extensively with RNase free water and dry it with a gas duster.
  3. Prepare a small box of ice containing 5x RT-mix and 20x DTT aliquots. Store the RTase and RNase inhibitor aliquots at -20 °C in a benchtop cooler.
  4. Transfer a slice into the recording chamber perfused at 1–2 mL/min with oxygenated aCSF.
  5. Pull patch pipettes (1–2 µm open tip diameter, 3–5 MΩ) from borosilicate glass while wearing gloves and fill one of them with 8 μL of internal solution. Keep in mind that the knobs of the pipette puller are a source of RNase contamination.
  6. Place the pipette in the pipette holder while wearing new gloves and not touching the filament with fingers.
  7. Approach the patch pipette with a positive pressure and perform whole-cell recording to characterize the targeted cell (Figure 4).
  8. In order to preserve the mRNAs, limit the time in whole-cell configuration to 20 min41.
  9. At the end of the recording, prepare a 500 µL PCR tube filled with 2 µL of 5x RT Mix and 0.5 µL of 20x DTT, spin it, and store it on ice.
  10. Harvest the cell cytoplasm by applying a gentle negative pressure (Figure 4). Visually control that the cell's content comes inside the pipette while maintaining a tight seal.
    1. Avoid as much as possible collecting the nucleus if some intron-less genes are considered. In such a case always include a set of primers aimed at amplifying gDNA to probe for genomic contamination.
    2. If the nucleus is coming close to the tip of the pipette, release the negative pressure and move the pipette away from the nucleus. Ensure that the tight seal is preserved and restart to collect the cytoplasm at the new pipette location.
  11. Stop the harvesting when no more material is coming and/or before losing the tight seal.
  12. Withdraw the pipette gently to form an outside-out patch to limit contamination by the extracellular debris (Figure 4) and to favor the closure of the cell membrane for subsequent histochemical analysis.
  13. Keep in mind that knobs, micromanipulators, computer keyboard, or computer mouse are potential sources of RNase contamination. If they have been touched during the recording, change gloves.
  14. Attach the pipette to the expeller and expel its content into the PCR tube by applying a positive pressure (Figure 1).
  15. Break the tip of the pipette into the PCR tube to help the collection of its content.
  16. Briefly centrifuge the tube, add 0.5 µL of RNase inhibitor and 0.5 µL of RTase, mix gently, centrifuge again, and incubate overnight at 37 °C.
  17. Spin the tube and store it at -80 °C for up to several months until PCR analysis.
  18. Perform the first amplification step directly in the tube containing the ~10 µL of RT products by adding water (q.s., 80 µL), 10 µL of 10x buffer, and 0.5 µL (2.5 U) of Taq polymerase. Then, follow the instructions described in steps 4.8.2–4.8.8.

8. Histochemical Staining of the Recorded Cell (Optional)

  1. Following the electrophysiological recordings, maintain the slice for 20 min in oxygenated aCSF to allow the diffusion of biocytin in the axonal and dendritic tree.
  2. Place the slice in a well of a 24-well plate and fix it overnight at 4 °C with 1 mL of 4% paraformaldehyde in 0.1 M Phosphate Buffer.
  3. Wash the slices 4 times, 5 min each, with 1 mL Phosphate Buffered Saline (PBS).
  4. In the same well, permeabilize and saturate the slice during 1 h at room temperature with 1 mL of PBS supplemented with 0.25% Triton X-100 and 0.2% gelatin from cold water fish skin (PBS-GT).
  5. Incubate overnight with a fluorescently labeled avidin diluted at 1/400 in 250–500 µL of PBS-GT.
  6. Wash 5 times, 5 min each, with 1 mL of PBS and mount the slices.

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Results

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,

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Discussion

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'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 µm open tip diamet...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
MACAW v.2.0.5NCBIMultiple alignement for primer design
DithiothreitolVWR443852ART
Random primersSigma-Aldrich (Merck)11034731001RT
dNTPsGE Healthcare Life Sciences28-4065-52RT and PCR
RNasin Ribonuclease InhibitorsPromegaN2511RT
SuperScript II Reverse TranscriptaseInvitrogen18064014RT
Taq DNA PolymeraseQiagen201205PCR
Mineral OilSigma-Aldrich (Merck)M5904-5MLPCR
PCR primersSigma-Aldrich (Merck)PCR / desalted and diluted at 200 µM
Tubes, 0.5 mL, flat capThermoFisher ScientificAB0350RT and PCR
BT10 Series - 10 µL Filter TipNeptune ScientificBT10RT and PCR
BT20 Series - 20 µL Filter TipNeptune ScientificBT20RT and PCR
BT200 Series - 200 µL Filter TipNeptune ScientificBT200RT and PCR
BT1000 Series - 1000 µL Filter TipNeptune ScientificBT1000.96RT and PCR
DNA Thermal CylcerPerkin Elmer CetusPCR
Ethidium BromideSigma-Aldrich (Merck)E1510-10MLAgarose gel electrophoresis
Tris-Borate-EDTA bufferSigma-Aldrich (Merck)T4415-1LAgarose gel electrophoresis
UltraPure AgaroseLife Technologies16500-500Agarose gel electrophoresis
ΦX174 DNA-Hae III DigestNEB (New England BioLabs)N3026SAgarose gel electrophoresis
EDA 290KodakAgarose gel electrophoresis
Electrophoresis Power supply EPS 3500Pharmacia BiotechAgarose gel electrophoresis
Midi Horizontal Elecrophoresis Unit Model SHU13Sigma-Aldrich (Merck)Agarose gel electrophoresis
Smooth paper with satin appearanceFisherbrand1748BPatch clamp internal solution
Potassium HydroxydeSigma-Aldrich (Merck)60377Patch clamp internal solution
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acidSigma-Aldrich (Merck)E3889Patch clamp internal solution
HEPESSigma-Aldrich (Merck)H4034Patch clamp internal solution
Potassium D-gluconateSigma-Aldrich (Merck)G4500Patch clamp internal solution
Magnesium chloride solutionSigma-Aldrich (Merck)M1028Patch clamp internal solution
5500 Vapor Pressure OsmometerWescorPatch clamp internal solution
BiocytinSigma-Aldrich (Merck)B4261Patch clamp internal solution
SucroseSigma-Aldrich (Merck)S5016Slice preparation
D-(+)-Glucose monohydrateSigma-Aldrich (Merck)49159Slice preparation
Sodium chlorideSigma-Aldrich (Merck)S6191Slice preparation
Potassium chlorideSigma-Aldrich (Merck)60128Slice preparation
Sodium bicarbonateSigma-Aldrich (Merck)31437-MSlice preparation
Sodium phosphate monobasicSigma-Aldrich (Merck)S5011Slice preparation
Magnesium chloride solutionSigma-Aldrich (Merck)63069Slice preparation
Calcium chloride solutionSigma-Aldrich (Merck)21115Slice preparation
Kynurenic acidSigma-Aldrich (Merck)K3375Slice preparation
IsofluranePiramal Healthcare UKSlice preparation
VT 1000SLeica Biosystems14047235613Slice preparation
Hydrogen peroxide solutionSigma-Aldrich (Merck)H1009Patch Clamp set-up cleaning
Thin Wall Glass Capillaries with filamentWorld Precision InstrumentsTW150F-4Patch Clamp
PP-83NarishigePatch Clamp
Eppendorf MicroloaderEppendorf5242956003Patch Clamp
BX51WI Upright microscopeOlympusPatch Clamp
XC-ST70/CE CCD B/W VIDEO CAMERASonyPatch Clamp
Axopatch 200B AmplifierMolecular DevicesPatch Clamp
Digidata 1440Molecular DevicesPatch Clamp
pCLAMP 10 software suiteMolecular DevicesPatch Clamp
10 mL syringeTerumoSS-10ESExpelling
E Series with Straight Body (Holder)Phymep64-0997Expelling
Sodium phosphate dibasicSigma-Aldrich (Merck)S7907Histochemical revelation
Sodium phosphate monobasicSigma-Aldrich (Merck)S8282Histochemical revelation
ParaformaldehydeSigma-Aldrich (Merck)P6148Histochemical revelation
Triton X-100Sigma-Aldrich (Merck)X100Histochemical revelation
Gelatin from cold water fish skinSigma-Aldrich (Merck)G7041Histochemical revelation
Streptavidin, Alexa Fluor 488 conjugateThermoFisher ScientificS11223Histochemical revelation
24-well plateGreiner Bio-One662160Histochemical revelation

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Single Cell RT PCRPatch clamp RecordingMultiplex PCRCytoplasm HarvestingGene Expression AnalysisHot Start PCRAgarose Gel ElectrophoresisBrain Slice PreparationElectrophysiological Recording
Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp
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