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.
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References
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Acknowledgments
This work is funded by the intramural research program at the National Institutes of Health. PC is an International Society for the Advancement of Cytometry Marylou Ingram Scholar.
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1 Electronic Supplementary Materials
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Supplemental File 1
Assay Database.xls (XLSX 127 kb)
Supplemental File 2
Assay Panel Worksheet.xls (XLSX 45 kb)
Supplemental File 3
FACS 0.2× Assay Mix and 20× Assay Plate.xls (XLSX 53 kb)
Supplemental File 4
FACS RT-PreAMP Reaction Mix.xls (XLS 42 kb)
Supplemental File 5
Sample Map.jmp (JMP 14 kb)
Supplemental File 6
Sample Map.xls (XLS 30 kb)
Supplemental File 7
Ct Data Raw.xls (XLS 1568 kb)
Supplemental File 8
FACS Mapping Script.jsl (JSL 4 kb)
Supplemental File 9
PROBE MAP.jmp (JMP 4 kb)
Supplemental File 10
SAMPLE MAP.jmp (JMP 14 kb)
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Dominguez, M., Roederer, M., Chattopadhyay, P.K. (2017). Highly Multiplexed, Single Cell Transcriptomic Analysis of T-Cells by Microfluidic PCR. In: Lugli, E. (eds) T-Cell Differentiation. Methods in Molecular Biology, vol 1514. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6548-9_15
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DOI: https://doi.org/10.1007/978-1-4939-6548-9_15
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6546-5
Online ISBN: 978-1-4939-6548-9
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