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. 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.affiliation: Biological Science and Technology, Sandia National Laboratories, MS 9292, 969, Livermore, CA, 94551-0969, USA.
authors.forename: Meiye
authors.initials: M
authors.lastname: Wu
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Piccini
authors.forename: Anup K
authors.initials: AK
authors.lastname: Singh
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D004867:Equipment Design; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation* / Q000379:methods; D019169:Jurkat Cells; D037181:Lectins, C-Type / Q000032:analysis; D035683:MicroRNAs / Q000032:analysis*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods; D009841:Oligonucleotides / Q000032:analysis*; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D037181:Lectins, C-Type; D035683:MicroRNAs; D009841:Oligonucleotides; D000099192:CD69 Antigens; C524942:MIRN155 microRNA, human; C477371:locked nucleic acid