Single Cell MicroRNA Analysis Using Microfluidic Flow Cytometry
Abstract
MicroRNAs (miRNAs) are non-coding small RNAs that have cell type and cell context-dependent expression and function. To study miRNAs at single-cell resolution, we have developed a novel microfluidic approach, where flow fluorescent in situ hybridization (flow-FISH) using locked-nucleic acid probes is combined with rolling circle amplification to detect the presence and localization of miRNA. Furthermore, our flow cytometry approach allows analysis of gene-products potentially targeted by miRNA together with the miRNA in the same cells. We demonstrate simultaneous measurement of miR155 and CD69 in 12-O-tetradecanoylphorbol 13-acetate (PMA) and Ionomycin stimulated Jurkat cells. The flow-FISH method can be completed in ,10 h, utilizes only 170 nL of reagent per experimental condition, and is the first to directly detect miRNA in single cells using flow cytometry.
- Authors:
-
- Sandia National Lab. (SNL-CA), Livermore, CA (United States). Dept. of Biotechnology and Bioengineering; Univ. of California, Berkeley, CA (United States). Biochemistry and Molecular Biology Graduate Group
- Sandia National Lab. (SNL-CA), Livermore, CA (United States). Dept. of Biotechnology and Bioengineering
- Univ. of California, Berkeley, CA (United States). Biochemistry and Molecular Biology Graduate Group; Univ. of California, Davis, CA (United States). Dept. of Hematology and Oncology
- Univ. of California, Berkeley, CA (United States). Biochemistry and Molecular Biology Graduate Group
- Publication Date:
- Research Org.:
- Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
- OSTI Identifier:
- 1627582
- Grant/Contract Number:
- AC04-94AL85000
- Resource Type:
- Accepted Manuscript
- Journal Name:
- PLoS ONE
- Additional Journal Information:
- Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 1932-6203
- Publisher:
- Public Library of Science
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Science & Technology - Other Topics
Citation Formats
Wu, Meiye, Piccini, Matthew, Koh, Chung-Yan, Lam, Kit S., and Singh, Anup K. Single Cell MicroRNA Analysis Using Microfluidic Flow Cytometry. United States: N. p., 2013.
Web. doi:10.1371/journal.pone.0055044.
Wu, Meiye, Piccini, Matthew, Koh, Chung-Yan, Lam, Kit S., & Singh, Anup K. Single Cell MicroRNA Analysis Using Microfluidic Flow Cytometry. United States. https://doi.org/10.1371/journal.pone.0055044
Wu, Meiye, Piccini, Matthew, Koh, Chung-Yan, Lam, Kit S., and Singh, Anup K. Wed .
"Single Cell MicroRNA Analysis Using Microfluidic Flow Cytometry". United States. https://doi.org/10.1371/journal.pone.0055044. https://www.osti.gov/servlets/purl/1627582.
@article{osti_1627582,
title = {Single Cell MicroRNA Analysis Using Microfluidic Flow Cytometry},
author = {Wu, Meiye and Piccini, Matthew and Koh, Chung-Yan and Lam, Kit S. and Singh, Anup K.},
abstractNote = {MicroRNAs (miRNAs) are non-coding small RNAs that have cell type and cell context-dependent expression and function. To study miRNAs at single-cell resolution, we have developed a novel microfluidic approach, where flow fluorescent in situ hybridization (flow-FISH) using locked-nucleic acid probes is combined with rolling circle amplification to detect the presence and localization of miRNA. Furthermore, our flow cytometry approach allows analysis of gene-products potentially targeted by miRNA together with the miRNA in the same cells. We demonstrate simultaneous measurement of miR155 and CD69 in 12-O-tetradecanoylphorbol 13-acetate (PMA) and Ionomycin stimulated Jurkat cells. The flow-FISH method can be completed in ,10 h, utilizes only 170 nL of reagent per experimental condition, and is the first to directly detect miRNA in single cells using flow cytometry.},
doi = {10.1371/journal.pone.0055044},
journal = {PLoS ONE},
number = 1,
volume = 8,
place = {United States},
year = {Wed Jan 30 00:00:00 EST 2013},
month = {Wed Jan 30 00:00:00 EST 2013}
}
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Works referenced in this record:
Microfluidic fluorescence in situ hybridization and flow cytometry (μFlowFISH)
journal, January 2011
- Liu, Peng; Meagher, Robert J.; Light, Yooli K.
- Lab on a Chip, Vol. 11, Issue 16, p. 2673-2679
Microfluidic fluorescence in situ hybridization and flow cytometry (μFlowFISH)
journal, January 2011
- Liu, Peng; Meagher, Robert J.; Light, Yooli K.
- Lab on a Chip, Vol. 11, Issue 16, p. 2673-2679
Proximity ligation assays for sensitive and specific protein analyses
journal, October 2005
- Gustafsdottir, Sigrun M.; Schallmeiner, Edith; Fredriksson, Simon
- Analytical Biochemistry, Vol. 345, Issue 1, p. 2-9
CD69 and Regulatiof the Immune Function
journal, January 1999
- Marzio, R.; Mauël, J.; Betz-Corradin, S.
- Immunopharmacology and Immunotoxicology, Vol. 21, Issue 3, p. 565-582
Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes
journal, December 2004
- Válóczi, Anna; Hornyik, Csaba; Varga, Nóra
- Nucleic Acids Research, Vol. 32, Issue 22, p. e175
Transcriptome and targetome analysis in MIR155 expressing cells using RNA-seq
journal, June 2010
- Xu, G.; Fewell, C.; Taylor, C.
- RNA, Vol. 16, Issue 8, p. 1610-1622
Single cell genome sequencing
journal, June 2012
- Yilmaz, Suzan; Singh, Anup K.
- Current Opinion in Biotechnology, Vol. 23, Issue 3, p. 437-443
CD69 and Regulatiof the Immune Function
journal, January 1999
- Marzio, R.; Mauël, J.; Betz-Corradin, S.
- Immunopharmacology and Immunotoxicology, Vol. 21, Issue 3, p. 565-582
Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines
journal, May 2012
- Powell, Ashley A.; Talasaz, AmirAli H.; Zhang, Haiyu
- PLoS ONE, Vol. 7, Issue 5, Article No. e33788
Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution
journal, January 2012
- Wu, Meiye; Perroud, Thomas D.; Srivastava, Nimisha
- Lab on a Chip, Vol. 12, Issue 16, p. 2823-2831
Cytokine detection by antibody-based proximity ligation
journal, May 2004
- Gullberg, M.; Gustafsdottir, S. M.; Schallmeiner, E.
- Proceedings of the National Academy of Sciences, Vol. 101, Issue 22, p. 8420-8424
CD69 Association with Jak3/Stat5 Proteins Regulates Th17 Cell Differentiation
journal, August 2010
- Martin, P.; Gomez, M.; Lamana, A.
- Molecular and Cellular Biology, Vol. 30, Issue 20, p. 4877-4889
MicroRNA In Situ Hybridization
book, December 2011
- Nielsen, Boye Schnack; Fan, Jian-Bing
- Next-Generation MicroRNA Expression Profiling Technology, p. 67-84
A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA)
journal, March 2006
- Castoldi, Mirco; Schmidt, Sabine; Benes, Vladimir
- RNA, Vol. 12, Issue 5, p. 913-920
A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA)
journal, March 2006
- Castoldi, Mirco; Schmidt, Sabine; Benes, Vladimir
- RNA, Vol. 12, Issue 5, p. 913-920
Single cell genome sequencing
journal, June 2012
- Yilmaz, Suzan; Singh, Anup K.
- Current Opinion in Biotechnology, Vol. 23, Issue 3, p. 437-443
MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy
journal, November 2011
- Kasinski, Andrea L.; Slack, Frank J.
- Nature Reviews Cancer, Vol. 11, Issue 12
Single-cell protein analysis
journal, February 2012
- Wu, Meiye; Singh, Anup K.
- Current Opinion in Biotechnology, Vol. 23, Issue 1, p. 83-88
Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution
journal, January 2012
- Wu, Meiye; Perroud, Thomas D.; Srivastava, Nimisha
- Lab on a Chip, Vol. 12, Issue 16, p. 2823-2831
Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment
journal, May 2010
- de Planell-Saguer, Mariàngels; Rodicio, María Celina; Mourelatos, Zissimos
- Nature Protocols, Vol. 5, Issue 6, p. 1061-1073
Cytokine detection by antibody-based proximity ligation
journal, May 2004
- Gullberg, M.; Gustafsdottir, S. M.; Schallmeiner, E.
- Proceedings of the National Academy of Sciences, Vol. 101, Issue 22, p. 8420-8424
Foxp3-Dependent MicroRNA155 Confers Competitive Fitness to Regulatory T Cells by Targeting SOCS1 Protein
journal, January 2009
- Lu, Li-Fan; Thai, To-Ha; Calado, Dinis Pedro
- Immunity, Vol. 30, Issue 1, p. 80-91
Regulation of the Germinal Center Response by MicroRNA-155
journal, April 2007
- Thai, T.-H.; Calado, D. P.; Casola, S.
- Science, Vol. 316, Issue 5824, p. 604-608
MicroRNA expression profiling of single whole embryonic stem cells
journal, January 2006
- Tang, Fuchou; Hajkova, Petra; Barton, Sheila C.
- Nucleic Acids Research, Vol. 34, Issue 2, p. e9-e9
miRNA in situ hybridization in formaldehyde and EDC–fixed tissues
journal, January 2009
- Pena, John T. G.; Sohn-Lee, Cherin; Rouhanifard, Sara H.
- Nature Methods, Vol. 6, Issue 2, p. 139-141
Absolute quantitation of microRNAs with a PCR-based assay
journal, December 2006
- Duncan, David D.; Eshoo, Mark; Esau, Christine
- Analytical Biochemistry, Vol. 359, Issue 2, p. 268-270
MicroRNAs in Sjögren's syndrome as a prototypic autoimmune disease
journal, July 2010
- Alevizos, Ilias; Illei, Gabor G.
- Autoimmunity Reviews, Vol. 9, Issue 9
A custom microarray platform for analysis of microRNA gene expression
text, January 2004
- Joel, Parker,; Scott, Hammond,; Michael, Thomson, J.
- The University of North Carolina at Chapel Hill University Libraries
MicroRNA expression profiling of single whole embryonic stem cells
journal, January 2006
- Tang, Fuchou; Hajkova, Petra; Barton, Sheila C.
- Nucleic Acids Research, Vol. 34, Issue 2, p. e9-e9
Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment
journal, May 2010
- de Planell-Saguer, Mariàngels; Rodicio, María Celina; Mourelatos, Zissimos
- Nature Protocols, Vol. 5, Issue 6, p. 1061-1073
MicroRNAs: new regulators of immune cell development and function
journal, July 2008
- Baltimore, David; Boldin, Mark P.; O'Connell, Ryan M.
- Nature Immunology, Vol. 9, Issue 8
MicroRNAs: new regulators of immune cell development and function
journal, July 2008
- Baltimore, David; Boldin, Mark P.; O'Connell, Ryan M.
- Nature Immunology, Vol. 9, Issue 8
Foxp3-Dependent MicroRNA155 Confers Competitive Fitness to Regulatory T Cells by Targeting SOCS1 Protein
journal, January 2009
- Lu, Li-Fan; Thai, To-Ha; Calado, Dinis Pedro
- Immunity, Vol. 30, Issue 1, p. 80-91
MicroRNA functions in animal development and human disease
journal, September 2005
- Alvarez-Garcia, I.
- Development, Vol. 132, Issue 21
Micromanagement of the immune system by microRNAs
journal, February 2008
- Lodish, Harvey F.; Zhou, Beiyan; Liu, Gwen
- Nature Reviews Immunology, Vol. 8, Issue 2
220-plex microRNA expression profile of a single cell
journal, September 2006
- Tang, Fuchou; Hajkova, Petra; Barton, Sheila C.
- Nature Protocols, Vol. 1, Issue 3, p. 1154-1159
A custom microarray platform for analysis of microRNA gene expression
journal, September 2004
- Thomson, J. Michael; Parker, Joel; Perou, Charles M.
- Nature Methods, Vol. 1, Issue 1, p. 47-53
MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy
journal, November 2011
- Kasinski, Andrea L.; Slack, Frank J.
- Nature Reviews Cancer, Vol. 11, Issue 12
220-plex microRNA expression profile of a single cell
journal, September 2006
- Tang, Fuchou; Hajkova, Petra; Barton, Sheila C.
- Nature Protocols, Vol. 1, Issue 3, p. 1154-1159
Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines
journal, May 2012
- Powell, Ashley A.; Talasaz, AmirAli H.; Zhang, Haiyu
- PLoS ONE, Vol. 7, Issue 5, Article No. e33788
The microRNA Registry
journal, January 2004
- Griffiths-Jones, S.
- Nucleic Acids Research, Vol. 32, Issue 90001
Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs
journal, November 2005
- Raymond, Christopher K.; Roberts, Brian S.; Garrett-Engele, Phillip
- RNA, Vol. 11, Issue 11, p. 1737-1744
MicroRNAs: A new class of regulatory genes affecting metabolism
journal, July 2006
- Krützfeldt, Jan; Stoffel, Markus
- Cell Metabolism, Vol. 4, Issue 1
Transcriptome and targetome analysis in MIR155 expressing cells using RNA-seq
journal, June 2010
- Xu, G.; Fewell, C.; Taylor, C.
- RNA, Vol. 16, Issue 8, p. 1610-1622
In Situ Proximity Ligation Assay for Microscopy and Flow Cytometry
journal, April 2011
- Leuchowius, Karl-Johan; Weibrecht, Irene; Söderberg, Ola
- Current Protocols in Cytometry, Vol. 56, Issue 1, p. 9.36.1-9.36.15
MicroRNA functions in animal development and human disease
journal, September 2005
- Alvarez-Garcia, I.
- Development, Vol. 132, Issue 21
The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occurring at a pre-translational level.
journal, July 1984
- Weiss, A.; Wiskocil, R. L.; Stobo, J. D.
- The Journal of Immunology, Vol. 133, Issue 1
Works referencing / citing this record:
Fluorescent analysis of bioactive molecules in single cells based on microfluidic chips
journal, January 2018
- Fan, Yuanyuan; Dong, Defang; Li, Qingling
- Lab on a Chip, Vol. 18, Issue 8
Microfluidic cytometric analysis of cancer cell transportability and invasiveness
journal, September 2015
- Liu, Zongbin; Lee, Yeonju; Jang, Joon hee
- Scientific Reports, Vol. 5, Issue 1
Microfluidic flow cytometry: The role of microfabrication methodologies, performance and functional specification
journal, March 2018
- Shrirao, Anil B.; Fritz, Zachary; Novik, Eric M.
- TECHNOLOGY, Vol. 06, Issue 01
Droplet microfluidics in (bio)chemical analysis
journal, January 2015
- Basova, Evgenia Yu; Foret, Frantisek
- The Analyst, Vol. 140, Issue 1
Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine
journal, January 2014
- Ali, M. Monsur; Li, Feng; Zhang, Zhiqing
- Chemical Society Reviews, Vol. 43, Issue 10
Microfluidic epigenomic mapping technologies for precision medicine
journal, January 2019
- Deng, Chengyu; Naler, Lynette B.; Lu, Chang
- Lab on a Chip, Vol. 19, Issue 16
Multiplexed PCR-Free Detection of MicroRNAs in Single Cancer Cells Using a DNA-Barcoded Microtrough Array Chip
journal, March 2019
- Wang, Nayi; Lu, Yao; Chen, Zhuo
- Micromachines, Vol. 10, Issue 4
Microfluidic Multiplexing in Bioanalyses
journal, October 2013
- Araz, M. Kursad; Tentori, Augusto M.; Herr, Amy E.
- Journal of Laboratory Automation, Vol. 18, Issue 5
Inverse relationship between microRNA-155 and -184 expression with increasing conjunctival inflammation during ocular Chlamydia trachomatis infection
journal, December 2015
- Derrick, Tamsyn; Last, Anna R.; Burr, Sarah E.
- BMC Infectious Diseases, Vol. 16, Issue 1
miRNA dysregulation in cancer: towards a mechanistic understanding
journal, March 2014
- Palanichamy, Jayanth Kumar; Rao, Dinesh S.
- Frontiers in Genetics, Vol. 5
Evaluation of microRNA-205 expression as a potential triage marker for patients with low-grade squamous intraepithelial lesions
journal, March 2017
- Xie, Hong; Norman, Ingrid; Hjerpe, Anders
- Oncology Letters, Vol. 13, Issue 5
Probing cytoplasmic and nuclear microRNAs in single living cells via plasmonic affinity sandwich assay
journal, January 2018
- Liu, Jia; Wen, Yanrong; He, Hui
- Chemical Science, Vol. 9, Issue 36
Inverse relationship between microRNA-155 and -184 expression with increasing conjunctival inflammation during ocular Chlamydia trachomatis infection
journal, December 2015
- Derrick, Tamsyn; Last, Anna R.; Burr, Sarah E.
- BMC Infectious Diseases, Vol. 16, Issue 1
Microfluidic cytometric analysis of cancer cell transportability and invasiveness
journal, September 2015
- Liu, Zongbin; Lee, Yeonju; Jang, Joon hee
- Scientific Reports, Vol. 5, Issue 1
miRNA dysregulation in cancer: towards a mechanistic understanding
journal, March 2014
- Palanichamy, Jayanth Kumar; Rao, Dinesh S.
- Frontiers in Genetics, Vol. 5