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Highly Multiplexed, Single Cell Transcriptomic Analysis of T-Cells by Microfluidic PCR.

Maria Dominguez1, Mario Roederer1, Pratip K Chattopadhyay2

  • 1Immunotechnology Section, Vaccine Research Center, NIH, 40 Convent Dr., Room 5612, Bethesda, MD, 20892, USA.

Methods in Molecular Biology (Clifton, N.J.)
|October 28, 2016
PubMed
Summary

Multiplexed single-cell gene expression analysis using Fluidigm

Keywords:
MicrofluidicQuantitative competitive real-time PCRSingle cell analysisT-cellTranscriptome

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Area of Science:

  • Molecular Biology
  • Immunology
  • Bioinformatics

Background:

  • Single-cell analysis allows for detailed study of cellular heterogeneity.
  • Measuring multiple mRNA transcripts simultaneously from single cells is a recent technological advancement.
  • Understanding T-cell heterogeneity is crucial for immune monitoring.

Purpose of the Study:

  • To describe methods and considerations for multiplexed single-cell gene expression analysis.
  • To detail the use of Fluidigm's BioMark platform for this purpose.
  • To highlight the application of this technology in characterizing T-cell heterogeneity.

Main Methods:

  • Qualification of primer/probes for multiplex assays.
  • Selection of genes for 96-parameter panels.
  • Performing reverse transcription and cDNA synthesis.
  • Operation of the Fluidigm BioMark instrument.
  • Data analysis strategies for single-cell gene expression data.

Main Results:

  • Established protocols for primer/probe qualification.
  • Guidelines for gene selection in high-parameter panels.
  • Detailed workflow for single-cell multiplex gene expression analysis.
  • Considerations for robust data analysis.

Conclusions:

  • The described methods enable reliable multiplexed single-cell gene expression profiling.
  • Fluidigm's BioMark platform is a valuable tool for this application.
  • This technology significantly aids in characterizing T-cell heterogeneity for immune monitoring.