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

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 types^1,2,3,4.

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 e....

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 followi....

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 interneurons^3,

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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