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

Maria Dominguez; Mario Roederer; Pratip K Chattopadhyay

doi:10.1007/978-1-4939-6548-9_15

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

Methods Mol Biol. 2017:1514:187-202. doi: 10.1007/978-1-4939-6548-9_15.

Authors

Maria Dominguez¹, Mario Roederer¹, Pratip K Chattopadhyay²

Affiliations

¹ Immunotechnology Section, Vaccine Research Center, NIH, 40 Convent Dr., Room 5612, Bethesda, MD, 20892, USA.
² Immunotechnology Section, Vaccine Research Center, NIH, 40 Convent Dr., Room 5612, Bethesda, MD, 20892, USA. pchattop@mail.nih.gov.

PMID: 27787802
DOI: 10.1007/978-1-4939-6548-9_15

Abstract

Recently, technologies have been developed to measure expression of 96 (or more) mRNA transcripts at once from a single cell. Here we describe methods and important considerations for use of Fluidigm's BioMark platform for multiplexed single cell gene expression. We describe how to qualify primer/probes, select genes to examine in 96-parameter panels, perform the reverse transcription/cDNA synthesis step, and operate the instrument. In addition, we describe data analysis considerations. This technology has enormous value for characterizing the heterogeneity of T-cells, thereby providing a useful tool for immune monitoring.

Keywords: Microfluidic; Quantitative competitive real-time PCR; Single cell analysis; T-cell; Transcriptome.

Publication types

Research Support, N.I.H., Intramural

MeSH terms

Gene Expression Profiling / methods
Humans
Microfluidics / methods*
Polymerase Chain Reaction / methods*
RNA, Messenger / immunology
Single-Cell Analysis / methods*
T-Lymphocytes / immunology
Transcriptome / genetics*
Transcriptome / immunology

Substances

RNA, Messenger