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

Wu, Meiye; Piccini, Matthew Ernest; Koh, Chung -Yan; Singh, Anup K.

doi:10.1007/978-1-4939-1459-3_20

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

Journal Article · Wed Aug 20 00:00:00 EDT 2014 · Methods in Molecular Biology

DOI:https://doi.org/10.1007/978-1-4939-1459-3_20· OSTI ID:1333852

Wu, Meiye ^[1]; Piccini, Matthew Ernest ^[1]; Koh, Chung -Yan ^[1]; Singh, Anup K. ^[1]

Sandia National Lab. (SNL-CA), Livermore, CA (United States)

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.

Research Organization:: Sandia National Laboratories (SNL-CA), Livermore, CA (United States)

Sponsoring Organization:: USDOE National Nuclear Security Administration (NNSA)

Grant/Contract Number:: AC04-94AL85000

OSTI ID:: 1333852

Report Number(s):: SAND--2014-2572J; 569686

Journal Information:: Methods in Molecular Biology, Journal Name: Methods in Molecular Biology Vol. 1211; ISSN 1064-3745

Publisher:: SpringerCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
FISH
flow cytometry
fluorescence in situ hybridization
locked nucleic acid
microRNA
microfluidics
multiplexing
rolling circle amplification
single-cell resolution

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

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References (14)

Cited By (2)

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