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

J Vis Exp. 2020 Mar 8:(157):10.3791/60612. doi: 10.3791/60612.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Video-Audio Media

MeSH terms

  • Animals
  • Dehydration
  • Disease Models, Animal
  • Fluorescent Antibody Technique
  • Gastrointestinal Microbiome / genetics
  • Gene Regulatory Networks
  • Laser Capture Microdissection / methods*
  • Male
  • Microfluidics*
  • Opioid-Related Disorders / diagnosis*
  • Opioid-Related Disorders / genetics*
  • Rats, Sprague-Dawley
  • Real-Time Polymerase Chain Reaction*
  • Single-Cell Analysis*
  • Systems Biology*
  • Transcription, Genetic
  • Transcriptome*