title: Highly Multiplexed, Single Cell Transcriptomic Analysis of T-Cells by Microfluidic PCR.
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.
authors: Immunotechnology Section, Vaccine Research Center, NIH, 40 Convent Dr., Room 5612, Bethesda, MD, 20892, USA.
authors: Maria
authors: M
authors: Dominguez
authors: Immunotechnology Section, Vaccine Research Center, NIH, 40 Convent Dr., Room 5612, Bethesda, MD, 20892, USA.
authors: Mario
authors: M
authors: Roederer
authors: Immunotechnology Section, Vaccine Research Center, NIH, 40 Convent Dr., Room 5612, Bethesda, MD, 20892, USA. pchattop@mail.nih.gov.
authors: Pratip K
authors: PK
authors: Chattopadhyay
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D044085:Microfluidics / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000276:immunology; D059010:Single-Cell Analysis / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology; D059467:Transcriptome / Q000235:genetics* / Q000276:immunology
chemical_list: D012333:RNA, Messenger
keywords: Microfluidic; Quantitative competitive real-time PCR; Single cell analysis; T-cell; Transcriptome