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Title: miRNA detection at single-cell resolution using microfluidic LNA flow-FISH

Abstract

Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155 and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.

Authors:
 [1];  [1];  [1];  [1]
  1. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Publication Date:
Research Org.:
Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1333852
Report Number(s):
SAND-2014-2572J
Journal ID: ISSN 1064-3745; 569686
Grant/Contract Number:  
AC04-94AL85000
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Methods in Molecular Biology
Additional Journal Information:
Journal Volume: 1211; Journal ID: ISSN 1064-3745
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; microRNA; locked nucleic acid; fluorescence in situ hybridization; FISH; flow cytometry; multiplexing; single-cell resolution; microfluidics; rolling circle amplification

Citation Formats

Wu, Meiye, Piccini, Matthew Ernest, Koh, Chung -Yan, and Singh, Anup K. miRNA detection at single-cell resolution using microfluidic LNA flow-FISH. United States: N. p., 2014. Web. doi:10.1007/978-1-4939-1459-3_20.
Wu, Meiye, Piccini, Matthew Ernest, Koh, Chung -Yan, & Singh, Anup K. miRNA detection at single-cell resolution using microfluidic LNA flow-FISH. United States. doi:10.1007/978-1-4939-1459-3_20.
Wu, Meiye, Piccini, Matthew Ernest, Koh, Chung -Yan, and Singh, Anup K. Wed . "miRNA detection at single-cell resolution using microfluidic LNA flow-FISH". United States. doi:10.1007/978-1-4939-1459-3_20. https://www.osti.gov/servlets/purl/1333852.
@article{osti_1333852,
title = {miRNA detection at single-cell resolution using microfluidic LNA flow-FISH},
author = {Wu, Meiye and Piccini, Matthew Ernest and Koh, Chung -Yan and Singh, Anup K.},
abstractNote = {Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155 and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.},
doi = {10.1007/978-1-4939-1459-3_20},
journal = {Methods in Molecular Biology},
issn = {1064-3745},
number = ,
volume = 1211,
place = {United States},
year = {2014},
month = {8}
}

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