Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence

Summary

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

Abstract

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular phenotype diversity. Single-cell experiments investigating the network dynamics of biological systems demonstrate cellular and tissue responses to various conditions at biologically meaningful resolution. Herein, we explain our methods for gathering single cells from anatomically specific locations and accurately measuring a subset of their gene expression profiles. We combine laser capture microdissection (LCM) with microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). We also use this microfluidic RT-qPCR platform to measure the microbial abundance of gut contents.

Introduction

Measuring the gene expression profiles of single cells has demonstrated extensive phenotypic heterogeneity within a tissue. This complexity has complicated our understanding of the biological networks that govern tissue function. Our group and others have explored this phenomenon in many tissues and conditions^1,2,3,4,5,6. These experiments not only suggest that regulation of gene expression networks underlie such heterogeneity, but also that single-cell resolution reveals ....

Protocol

This study was carried out in accordance with the recommendations of Animal Care and Use Committee (IACUC) of Thomas Jefferson University and Drexel University College of Medicine. The protocol was approved by the Thomas Jefferson University and Drexel University College of Medicine IACUC.

1. Animal model

1. Insert two 75 mg slow-release morphine sulfate pellets or two placebo pellets subcutaneously in adult male Sprague-Dawley rats.
   1. Use a gown and gloves appropriately for minor sterile surgery. Shave the rat dorsum with clippers if necessary.
   2. Apply vet ointment to the animal's eyes. Anesthetize the rat w....

Results

The selection of the single cells was validated both visually and molecularly. Visually, cellular morphology was viewed before cell collection. Cells collected were then viewed at the QC station and the cellular nuclei stain (DAPI) overlapped with the single cell selection marker fluorescence. Figure 2A shows representative images of a slide with hemisected rat forebrain containing the CeA. Subsequent images (Figure 2

Discussion

Single-cell biology has demonstrated the heterogeneity of cellular phenotypes and robustness of tissue function. These findings have provided insight into the organization of biological systems at both macro and micro scales. Here, we describe the combination of two methods, LCM and microfluidic qPCR, to obtain single-cell transcriptome measures that provide anatomic specificity and transcriptional accuracy at a relatively low cost (Figure 1). Our group takes a systems biology approach and o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20X DNA Binding Dye	Fluidigm	100-7609	NA
2x GE Assay Loading Reagent	Fluidigm	85000802-R	NA
48.48 Dynamic Array IFC for Gene Expression	Fluidigm	BMK-M-48.48	NA
96.96 Dynamic Array IFC for Gene Expression	Fluidigm	BMK-M-96.96	NA
Anti-Cd11β Antibody	Genway Biotech	CCEC48	Microglia Stain
Anti-NeuN Antibody, clone A60	EMD Millipore	MAB377	Neuronal Stain
ArcturusXT Laser Capture Microdissection System	Arcturus	NA	NA
Biomark HD	Fluidigm	NA	RT-qPCR platform
Bovine Serum Antigen	Sigma-Aldrich	B4287
CapSure Macro LCM Caps	ThermoFisher Scientific	LCM0211	NA
CellDirect One-Step qRT-PCR Kit	ThermoFisher Scientific	11753500	Lysis buffer solution components
DAPI	ThermoFisher Scientific	62248	Nucleus Stain
DNA Suspension Buffer	TEKnova	T0221
Exonuclease I	New Englnad BioLabs, Inc.	M0293S	NA
ExtracSure Sample Extraction Device	ThermoFisher Scientific	LCM0208	NA
Fisherbrand Superfrost Plus Microscope Slides	ThermoFisher Scientific	22-037-246	Plain glass slides
GeneAmp Thin-Walled Reaction Tube	ThermoFisher Scientific	N8010611
GFAP Monoclonal Antibody	ThermoFisher Scientific	A-21294	Astrocyte Stain
Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488	ThermoFisher Scientific	A28175	Seconadry Antibody
IFC Controller	Fluidigm	NA	NA
RNaseOut	ThermoFisher Scientific	10777019
SsoFast EvaGreen Supermix with Low Rox	Bio-Rad	PN 172-5211	Rox master mix
SuperScript VILO cDNA Synthesis Kit	ThermoFisher Scientific	11754250	Contains VILO and SuperScript
T4 Gene 32 Protein	New Englnad BioLabs, Inc.	M0300S	NA
TaqMan PreAmp Master Mix	ThermoFisher Scientific	4391128	NA
TE Buffer	TEKnova	T0225	NA