Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

Wu, Meiye; Singh, Anup K.

doi:10.1177/2211068214542247

Title: Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

Full Record
Other Related Research

Abstract

In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.

Authors:

Wu, Meiye ^[1]; Singh, Anup K. ^[1]

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

Publication Date:: Tue Jul 15 00:00:00 EDT 2014

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

Sponsoring Org.:: USDOE National Nuclear Security Administration (NNSA)

OSTI Identifier:: 1235251

Report Number(s):: SAND-2014-2840J
Journal ID: ISSN 2211-0682; 569594

Grant/Contract Number:: AC04-94AL85000

Resource Type:: Accepted Manuscript

Journal Name:: Journal of Laboratory Automation

Additional Journal Information:: Journal Volume: 19; Journal Issue: 6; Journal ID: ISSN 2211-0682

Publisher:: SAGE

Country of Publication:: United States

Language:: English

Subject:: 42 ENGINEERING; protein; mRNA; microRNA; PTM; multiplex; single cell; microfluidics; flow cytometry; imaging

Citation Formats


                    Wu, Meiye, and Singh, Anup K. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution.  United States: N. p., 2014. 
Web.  doi:10.1177/2211068214542247.

Copy to clipboard


                    Wu, Meiye, & Singh, Anup K. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution.  United States.  https://doi.org/10.1177/2211068214542247

Copy to clipboard


                    Wu, Meiye, and Singh, Anup K. Tue .  
"Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution".  United States.  https://doi.org/10.1177/2211068214542247.  https://www.osti.gov/servlets/purl/1235251.

Copy to clipboard


                    
@article{osti_1235251,

  title        = {Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution},

  author       = {Wu, Meiye and Singh, Anup K.},

  abstractNote = {In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.},

  doi          = {10.1177/2211068214542247},

  journal      = {Journal of Laboratory Automation},

  number       = 6,

  volume       = 19,

  place        = {United States},

  year         = {Tue Jul 15 00:00:00 EDT 2014},

  month        = {Tue Jul 15 00:00:00 EDT 2014}

}

Copy to clipboard

Journal Article:

Free Publicly Available Full Text

Accepted Manuscript (DOE)

Publisher's Version of Record

https://doi.org/10.1177/2211068214542247

Other availability

Search WorldCat to find libraries that may hold this journal

Citation Metrics:

Cited by: 2 works

Citation information provided by
Web of Science

Save / Share:

Export Metadata

Save to My Library

Similar Records in DOE PAGES and OSTI.GOV collections:

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

Journal Article Wu, Meiye ; Piccini, Matthew Ernest ; Koh, Chung -Yan ; ... - Methods in Molecular Biology

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 miR155more »« less
Cited by 6
https://doi.org/10.1007/978-1-4939-1459-3_20

Full Text Available
Next-generation sequencing-based miRNA expression analysis in Parp1-deficient embryonic stem cell-derived exosomes

Journal Article Nozaki, Tadashige ; Sasaki, Yuka ; Division of Cellular Signaling, Laboratory of Collaborative Research, National Cancer Center Research Institute ; ... - Biochemical and Biophysical Research Communications

Highlights: • This is the first report for the extracellular role of Parp1 via exosomal miRNAs. • Parp1{sup −/−} embryonic stem cell-derived exosomes were examined for miRNA regulation. • Next-generation sequencing revealed upregulated and downregulated miRNAs. • Upregulated miRNAs were associated with five signaling pathways. • Gene ontology annotation for growth/stress-related signaling and cell communication. Poly (ADP-ribose) polymerase family, member 1 (Parp1) has pleiotropic and disparate functions in multiple cellular signaling pathways through post-translational protein modification. It contributes to the regulation of various cellular processes, including DNA damage repair, cell death, and cell differentiation, genetically or epigenetically. Meanwhile, the functionsmore »« less
https://doi.org/10.1016/J.BBRC.2018.03.073
Single Cell MicroRNA Analysis Using Microfluidic Flow Cytometry

Journal Article Wu, Meiye ; Piccini, Matthew ; Koh, Chung-Yan ; ... - PLoS ONE

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 ,10more »« less
https://doi.org/10.1371/journal.pone.0055044

Full Text Available
Hybridization selection of nucleic acid-protein complexes. 1. Detection of proteins cross-linked to specific mRNAs and DNA sequences by irradiation of intact Escherichia coli cells with ultraviolet light

Journal Article Schouten, J P - J. Biol. Chem.; (United States)

A method is presented for the detection in crude lysates of subnanogram amounts of proteins covalently bound to a specific nucleic acid sequence. The sensitivity of this method enabled the authors to study proteins cross-linked to specific DNA and mRNA sequences by irradiation of intact Escherichia coli cells with ultraviolet light. Among the proteins cross-linked to pBR322 DNA, the single strand binding protein, the HU-proteins, and the RNA polymerase beta and sigma subunits were present. Some, but not all proteins were cross-linked to 5-bromodeoxyuridine-substituted DNA more efficiently than to normal DNA. Ribosomal protein S1 is by far the most prominentmore »« less
Microfluidic platform for multiplexed detection in single cells and methods thereof

Patent Wu, Meiye ; Singh, Anup K.

The present invention relates to a microfluidic device and platform configured to conduct multiplexed analysis within the device. In particular, the device allows multiple targets to be detected on a single-cell level. Also provided are methods of performing multiplexed analyses to detect one or more target nucleic acids, proteins, and post-translational modifications.
Full Text Available

Similar Records