Advertisement

Microfluidic Approaches to Fluorescence In Situ Hybridization (FISH) for Detecting RNA Targets in Single Cells

  • Robert J. Meagher
  • Meiye Wu
Chapter

Abstract

Fluorescence in situ hybridization (FISH) is a powerful molecular technique in cell biology and microbiology for detection and localization of a nucleic acid target within an intact cell or chromosome spread, based on hybridization of a fluorescently labeled nucleic acid “probe” to its complementary target. In some instances, FISH analysis is performed on intact samples—whether thin tissue sections, or environmental samples, allowing the nucleic acid target to be localized in context with other cells. FISH evolved from in situ hybridization (ISH) techniques utilizing radiolabeled probes. By comparison, FISH typically utilizes small-molecule fluorophores. This labeling approach eliminates the hazards associated with radioactivity, and allows analysis with common laboratory instrumentation, including epifluorescence or laser-scanning confocal microscopes, or flow cytometers. Unlike PCR, sequencing, or most other nucleic acid analysis methods, FISH is fundamentally a single-cell measurement technique. Whether using imaging or flow cytometry as a readout, the signals from FISH are detected and analyzed on a cell-by-cell basis, affording a unique capability for studying rare events or heterogeneity within a population. Coupling of FISH with flow sorters also affords a unique capability for enriching cell or chromosome populations or even isolating single cells based on presence of specific nucleic acid targets [7, 10, 43].

Keywords

Lab on a chip Molecular diagnostics Gene expression Genetic analysis Single-cell analysis Nucleic acid hybridization Flow cytometry 

Notes

Acknowledgments

The authors acknowledge support from Sandia National Laboratories’ Laboratory Directed Research and Development (LDRD) program. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the US Department of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000.

References

  1. 1.
    Amann R, Fuchs BM (2008) Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Micro 6:339–348CrossRefGoogle Scholar
  2. 2.
    Baerlocher GM, Vulto I, de Jong G, Lansdorp PM (2006) Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protocols 1:2365–2376CrossRefPubMedGoogle Scholar
  3. 3.
    Chen CH, Cho SH, Tsai F, Erten A, Lo Y-H (2009) Microfluidic cell sorter with integrated piezoelectric actuator. Biomed Microdevices 11:1223–1231CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Crosetto N, Bienko M, van Oudenaarden A (2015) Spatially resolved transcriptomics and beyond. Nat Rev Genet 16:57–66CrossRefPubMedGoogle Scholar
  5. 5.
    Fu AY, Chou HP, Spence C, Arnold FH, Quake SR (2002) An integrated microfabricated cell sorter. Anal Chem 74:2451–2457CrossRefPubMedGoogle Scholar
  6. 6.
    Gerdts G, Luedke G (2006) FISH and chips: marine bacterial communities analyzed by flow cytometry based on microfluidics. J Microbiol Methods 64:232–240CrossRefPubMedGoogle Scholar
  7. 7.
    Giorgi D, Farina A, Grosso V, Gennaro A, Ceoloni C, Lucretti S (2013) FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. PLoS One 8:e57994CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gregori G, Patsekin V, Rajwa B, Jones J, Ragheb K, Holdman C, Robinson JP (2012) Hyperspectral cytometry at the single-cell level using a 32-channel photodetector. Cytometry A 81:35–44CrossRefPubMedGoogle Scholar
  9. 9.
    Itzkovitz S, van Oudenaarden A (2011) Validating transcripts with probes and imaging technology. Nat Methods 8:S12–S19CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kalyuzhnaya MG, Zabinsky R, Bowerman S, Baker DR, Lidstrom ME, Chistoserdova L (2006) Fluorescence in situ hybridization-flow cytometry-cell sorting-based method for separation and enrichment of type I and type II methanotroph populations. Appl Environ Microbiol 72:4293–4301CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Lantz AW, Brehm-Stecher BF, Armstrong DW (2008) Combined capillary electrophoresis and DNA-fluorescence in situ hybridization for rapid molecular identification of Salmonella Typhimurium in mixed culture. Electrophoresis 29:2477–2484CrossRefPubMedGoogle Scholar
  12. 12.
    Larsson C, Grundberg I, Soderberg O, Nilsson M (2010) In situ detection and genotyping of individual mRNA molecules. Nat Meth 7:395–397CrossRefGoogle Scholar
  13. 13.
    Latorra D, Campbell K, Wolter A, Hurley JM (2003) Enhanced allele-specific PCR discrimination in SNP genotyping using 3′ locked nucleic acid (LNA) primers. Hum Mutat 22:79–85CrossRefPubMedGoogle Scholar
  14. 14.
    Lebaron P, Catala P, Fajon C, Joux F, Baudart J, Bernard L (1997) A new sensitive, whole-cell hybridization technique for detection of bacteria involving a biotinylated oligonucleotide probe targeting rRNA and tyramide signal amplification. Appl Environ Microbiol 63:3274–3278PubMedPubMedCentralGoogle Scholar
  15. 15.
    Liu P, Meagher RJ, Light YK, Yilmaz S, Chakraborty R, Arkin AP, Hazen TC, Singh AK (2011) Microfluidic fluorescence in situ hybridization and flow cytometry (mu FlowFISH). Lab Chip 11:2673–2679CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Mao X, Nawaz AA, Lin S-CS, Lapsley MI, Zhao Y, McCoy JP, El-Deiry WS, Huang TJ (2012) An integrated, multiparametric flow cytometry chip using “microfluidic drifting” based three-dimensional hydrodynamic focusing. Biomicrofluidics 6:024113-024113-024119Google Scholar
  17. 17.
    Martel JM, Toner M (2013) Particle focusing in curved microfluidic channels. Sci Rep 3:3340CrossRefGoogle Scholar
  18. 18.
    Maruyama F, Kenzaka T, Yamaguchi N, Tani K, Nasu M (2003) Detection of bacteria carrying the stx(2) gene by in situ loop-mediated isothermal amplification. Appl Environ Microbiol 69:5023–5028CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Nelson PT, Baldwin DA, Kloosterman WP, Kauppinen S, Plasterk RH, Mourelatos Z (2006) RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain. RNA 12:187–191CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Nolan JP, Condello D, Duggan E, Naivar M, Novo D (2013) Visible and near infrared fluorescence spectral flow cytometry. Cytometry A 83:253–264CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Oliveira K, Procop GW, Wilson D, Coull J, Stender H (2002) Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes. J Clin Microbiol 40:247–251CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Packard MM, Shusteff M, Alocilja EC (2012) Microfluidic-based amplification-free bacterial DNA detection by dielectrophoretic concentration and fluorescent resonance energy transfer assisted in situ hybridization (FRET-ISH) (†,‡). Biosensors 2:405–416CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Pernthaler A, Amann R (2004) Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria. Appl Environ Microbiol 70:5426–5433CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Perroud TD, Kaiser JN, Sy JC, Lane TW, Branda CS, Singh AK, Patel KD (2008) Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces. Anal Chem 80:6365–6372CrossRefPubMedGoogle Scholar
  25. 25.
    Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5:877–879CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Rhee M, Valencia PM, Rodriguez MI, Langer R, Farokhzad OC, Karnik R (2011) Synthesis of size-tunable polymeric nanoparticles enabled by 3D hydrodynamic flow focusing in single-layer microchannels. Adv Mater 23:H79–H83CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Robertson KL, Verhoeven AB, Thach DC, Chang EL (2010) Monitoring viral RNA in infected cells with LNA flow-FISH. RNA 16:1679–1685CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Salimnia H, Fairfax MR, Lephart P, Morgan M, Gilbreath JJ, Butler-Wu SM, Templeton KE, Hamilton FJ, Wu F, Buckner R, Fuller D, Davis TE, Abdelhamed AM, Jacobs MR, Miller A, Pfrommer B, Carroll KC (2014) An international, prospective, multicenter evaluation of the combination of AdvanDx Staphylococcus QuickFISH BC with mecA XpressFISH for detection of methicillin-resistant Staphylococcus aureus isolates from positive blood cultures. J Clin Microbiol 52:3928–3932CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Schmidt KS, Borkowski S, Kurreck J, Stephens AW, Bald R, Hecht M, Friebe M, Dinkelborg L, Erdmann VA (2004) Application of locked nucleic acids to improve aptamer in vivo stability and targeting function. Nucleic Acids Res 32:5757–5765CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Shapiro HM (2003) Practical flow cytometry, 4th edn. Wiley, Hoboken, NJCrossRefGoogle Scholar
  31. 31.
    Silahtaroglu A, Pfundheller H, Koshkin A, Tommerup N, Kauppinen S (2004) LNA-modified oligonucleotides are highly efficient as FISH probes. Cytogenet Genome Res 107:32–37CrossRefPubMedGoogle Scholar
  32. 32.
    Simonnet C, Groisman A (2005) Two-dimensional hydrodynamic focusing in a simple microfluidic device. Appl Phys Lett 87:114104CrossRefGoogle Scholar
  33. 33.
    Skinner SO, Sepúlveda LA, Xu H, Golding I (2013) Measuring mRNA copy-number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization (smFISH). Nat Protoc 8:1100–1113CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Song Y, Peng R, Wang J, Pan X, Sun Y, Li D (2013) Automatic particle detection and sorting in an electrokinetic microfluidic chip. Electrophoresis 34:684–690CrossRefPubMedGoogle Scholar
  35. 35.
    Spurgeon SL, Jones RC, Ramakrishnan R (2008) High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS One 3:e1662CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Urbanek M, Nawrocka A, Krzyzosiak W (2015) Small RNA detection by in situ hybridization methods. Int J Mol Sci 16:13259CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Valm AM, Welch JLM, Rieken CW, Hasegawa Y, Sogin ML, Oldenbourg R, Dewhirst FE, Borisy GG (2011) Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging. Proc Natl Acad Sci 108:4152–4157CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Vester B, Wengel J (2004) LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43:13233–13241CrossRefPubMedGoogle Scholar
  39. 39.
    Warren L, Bryder D, Weissman IL, Quake SR (2006) Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Natl Acad Sci U S A 103:17807–17812CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Wu M, Piccini M, Koh CY, Lam KS, Singh AK (2013) Single cell microRNA analysis using microfluidic flow cytometry. PLoS One 8:e55044CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Wu M, Singh AK (2014) Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and posttranslational modifications at single-cell resolution. J Lab Autom 19:587–592CrossRefPubMedGoogle Scholar
  42. 42.
    Yamaguchi N, Ohba H, Nasu M (2006) Simple detection of small amounts of Pseudomonas cells in milk by using a microfluidic device. Lett Appl Microbiol 43:631–636CrossRefPubMedGoogle Scholar
  43. 43.
    Yilmaz S, Haroon MF, Rabkin BA, Tyson GW, Hugenholtz P (2010) Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations. ISME J 4:1352–1356CrossRefPubMedGoogle Scholar
  44. 44.
    Zhang Q, Zhu L, Feng H, Ang S, Chau FS, Liu W-T (2006) Microbial detection in microfluidic devices through dual staining of quantum dots-labeled immunoassay and RNA hybridization. Anal Chim Acta 556:171–177CrossRefPubMedGoogle Scholar
  45. 45.
    Zwirglmaier K (2005) Fluorescence in situ hybridisation (FISH)—the next generation. FEMS Microbiol Lett 246:151–158CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.Biotechnology and Bioengineering DepartmentSandia National LaboratoriesLivermoreUSA

Personalised recommendations