Skip to main content
SpringerLink
Log in

Multiplexed hydrogel microparticle suspension arrays for facile ribosomal RNA integrity assays

  • Research Paper
  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

Reliable RNA integrity assay is important for a wide range of applications in genomics and diagnostics, yet the existing technologies have certain limitations such as large amount of sample required, high cost of equipment and/or long turnaround times. We report a simple assay method to analyze bacterial ribosomal RNA (rRNA) from complex total RNA samples utilizing shape-encoded and single-stranded DNA-conjugated hydrogel microparticle suspension arrays with no need for target amplification and under standard fluorescence imaging conditions. We show that our simple microparticle-based sensing scheme is reliable, sequence-specific and presents a responsive binding behavior to target RNA concentrations. Moreover, the relative stability of 16S and 23S rRNA can be assessed in a simple shape encoding-based multiplexed format. Combined, these findings represent a significant step toward cheap, fast, simple, and reliable assays for the analysis of rRNA and general RNA integrity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Fleige, S. and M. W. Pfaffl (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Aspects Med. 27: 126–139.

    Article  CAS  Google Scholar 

  2. Ramaswamy, S. and T. R. Golub (2002) DNA microarrays in clinical oncology. J. Clin. Oncol. 20: 1932–1941.

    CAS  Google Scholar 

  3. Copois, V., F. Bibeau, C. Bascoul-Mollevi, N. Salvetat, P. Chalbos, C. Bareil, L. Candeil, C. Fraslon, E. Conseiller, V. Granci, P. Mazière, A. Kramar, M. Ychou, B. Pau, P. Martineau, F. Molina, and M. Del Rio (2007) Impact of RNA degradation on gene expression profiles: Assessment of different methods to reliably determine RNA quality. J. Biotechnol. 127: 549–559.

    Article  CAS  Google Scholar 

  4. Vermeulen, J., K. De Preter, S. Lefever, J. Nuytens, F. De Vloed, S. Derveaux, J. Hellemans, F. Speleman, and J. Vandesompele (2011) Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res. 39: e63.

    Article  CAS  Google Scholar 

  5. Gopinath, S. C. B., T.-H. Tang, Y. Chen, M. Citartan, and T. Lakshmipriya (2014) Bacterial detection: From microscope to smartphone. Biosens. Bioelectron. 60: 332–342.

    Article  CAS  Google Scholar 

  6. Ares, M. (2012) Bacterial RNA Isolation. Cold Spring Harb. Protoc. doi:10.1101/pdb.prot071068.

    Google Scholar 

  7. Thatcher, S. A. (2015) DNA/RNA preparation for molecular detection. Clin. Chem. 61: 89–99.

    Article  CAS  Google Scholar 

  8. Becker, C., A. Hammerle-Fickinger, I. Riedmaier, and M. W. Pfaffl (2010) mRNA and microRNA quality control for RTqPCR analysis. Methods 50: 237–243.

    Article  CAS  Google Scholar 

  9. Die, J. V. and B. Román (2012) RNA quality assessment: A view from plant qPCR studies. J. Exp. Bot. 63: 6069–6077.

    Article  CAS  Google Scholar 

  10. Manchester, K. L. (1996) Use of UV methods for measurement of protein and nucleic acid concentrations. Biotechniques 20: 968–970.

    CAS  Google Scholar 

  11. Sambrook, J. and D. Russell (2001) Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

    Google Scholar 

  12. Imbeaud, S., E. Graudens, V. Boulanger, X. Barlet, P. Zaborski, E. Eveno, O. Mueller, A. Schroeder, and C. Auffray (2005) Towards standardization of RNA quality assessment using userindependent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 33: e56.

    Article  Google Scholar 

  13. Schroeder, A., O. Mueller, S. Stocker, R. Salowsky, M. Leiber, M. Gassmann, S. Lightfoot, W. Menzel, M. Granzow, and T. Ragg (2006) The RIN: An RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 7: 3.

    Article  Google Scholar 

  14. Denisov, V., W. Strong, M. Walder, J. Gingrich, and H. Wintz. (2008) Development and validation of RQI: An RNA quality indicator for the Experion™ automated electrophoresis system. In: Tech Note. Bio-Rad.

    Google Scholar 

  15. Breadmore, M. C. (2012) Capillary and microchip electrophoresis: Challenging the common conceptions. J. Chromatogr. A 1221: 42–55.

    Article  CAS  Google Scholar 

  16. Duy, J., R. L. Smith, S. D. Collins, and L. B. Connell (2014) A field-deployable colorimetric bioassay for the rapid and specific detection of ribosomal RNA. Biosens. Bioelectron. 52: 433–437.

    Article  CAS  Google Scholar 

  17. Aslan, K., J. Huang, G. M. Wilson, and C. D. Geddes (2006) Metal-enhanced Fluorescence-based RNA sensing. J. Am. Chem. Soc. 128: 4206–4207.

    Article  CAS  Google Scholar 

  18. Foudeh, A. M., J. T. Daoud, S. P. Faucher, T. Veres, and M. Tabrizian (2014) Sub-femtomole detection of 16s rRNA from Legionella pneumophila using surface plasmon resonance imaging. Biosens. Bioelectron. 52: 129–135.

    Article  CAS  Google Scholar 

  19. Auer, H., S. Lyianarachchi, D. Newsom, M. I. Klisovic, U. Marcucci, and K. Kornacker (2003) Chipping away at the chip bias: RNA degradation in microarray analysis. Nat. Genet. 35: 292–293.

    Article  CAS  Google Scholar 

  20. Wilkes, T., A. Devonshire, S. Ellison, and C. Foy (2010) Evaluation of a novel approach for the measurement of RNA quality. BMC Res. Notes 3: 89.

    Article  Google Scholar 

  21. Tjong, V., H. Yu, A. Hucknall, and A. Chilkoti (2013) Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization. Anal. Chem. 85: 426–433.

    Article  CAS  Google Scholar 

  22. Squires, T. M., R. J. Messinger, and S. R. Manalis (2008) Making it stick: Convection, reaction and diffusion in surface-based biosensors. Nat. Biotechnol. 26: 417–426.

    Article  CAS  Google Scholar 

  23. Feldman, A. L., N. G. Costouros, E. Wang, M. Qian, F. M. Marincola, H. R. Alexander, and S. K. Libutti (2002) Advantages of mRNA amplification for microarray analysis. Biotechniques 33: 906–912, 914.

    CAS  Google Scholar 

  24. Pregibon, D. C., M. Toner, and P. S. Doyle (2007) Multifunctional encoded particles for high-throughput biomolecule analysis. Science 315: 1393–1396.

    Article  CAS  Google Scholar 

  25. Choi, N. W., J. Kim, S. C. Chapin, T. Duong, E. Donohue, P. Pandey, W. Broom, W. A. Hill, and P. S. Doyle (2012) Multiplexed Detection of mRNA using porosity-tuned hydrogel microparticles. Anal. Chem. 84: 9370–9378.

    CAS  Google Scholar 

  26. Rehman, F. N., M. Audeh, E. S. Abrams, P. W. Hammond, M. Kenney, and T. C. Boles (1999) Immobilization of acrylamidemodified oligonucleotides by co-polymerization. Nucleic Acids Res. 27: 649–655.

    Article  CAS  Google Scholar 

  27. Lewis, C. L., C.-H. Choi, Y. Lin, C.-S. Lee, and H. Yi (2010) Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays. Anal. Chem. 82: 5851–5858.

    Article  CAS  Google Scholar 

  28. Fuchs, B. M., K. Syutsubo, W. Ludwig, and R. Amann (2001) In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 67: 961–968.

    Article  CAS  Google Scholar 

  29. Nelson, B. P., M. R. Liles, K. B. Frederick, R. M. Corn, and R. M. Goodman (2002) Label-free detection of 16S ribosomal RNA hybridization on reusable DNA arrays using surface plasmon resonance imaging. Environ. Microbiol. 4: 735–743.

    Article  CAS  Google Scholar 

  30. Fuchs, B. M., G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann (1998) Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 64: 4973–4982.

    CAS  Google Scholar 

  31. Chapin, S. C., D. C. Pregibon, and P. S. Doyle (2009) Highthroughput flow alignment of barcoded hydrogel microparticles. Lab Chip 9: 3100–3109.

    Article  CAS  Google Scholar 

  32. Pregibon, D. C. and P. S. Doyle (2009) Optimization of Encoded Hydrogel Particles for Nucleic Acid Quantification. Anal. Chem. 81: 4873–4881.

    Article  CAS  Google Scholar 

  33. Jung, S. and H. Yi (2013) Facile strategy for protein conjugation with chitosan-poly(ethylene glycol) hybrid microparticle platforms via strain-promoted Alkyne–Azide Cycloaddition (SPAAC) reaction. Biomacromol. 14: 3892–3902.

    Article  CAS  Google Scholar 

  34. Carlsson, C., M. Jonsson, and B. Akerman (1995) Double bands in DNA gel electrophoresis caused by bis-intercalating dyes. Nucleic Acids Res. 23: 2413–2420.

    Article  CAS  Google Scholar 

  35. Rasband, W. S. (1997–2014) ImageJ. Bethesda U S National Institutes of Health, Maryland, USA. http://imagej.nih.gov/ij/.

    Google Scholar 

  36. Tam, M. F., J. A. Dodd, and W. E. Hill (1981) Physical characteristics of 16 S rRNA under reconstitution conditions. J. Biol. Chem. 256: 6430–6434.

    CAS  Google Scholar 

  37. Yoffe, A. M., P. Prinsen, A. Gopal, C. M. Knobler, W. M. Gelbart, and A. Ben-Shaul (2008) Predicting the sizes of large RNA molecules. Proc. Natl. Acad. Sci. U. S. A. 105: 16153–16158.

    Article  CAS  Google Scholar 

  38. Mellott, M. B., K. Searcy, and M. V. Pishko (2001) Release of protein from highly cross-linked hydrogels of poly(ethylene gly col) diacrylate fabricated by UV polymerization. Biomaterials 22: 929–941.

    Article  CAS  Google Scholar 

  39. Chiu, Y. C., E. Brey, and V. Pérez-Luna (2012) A study of the intrinsic autofluorescence of Poly (ethylene glycol)-co-(L-Lactic acid) Diacrylate. J. Fluoresc. 22: 907–913.

    Article  CAS  Google Scholar 

  40. Lee, A. G., C. P. Arena, D. J. Beebe, and S. P. Palecek (2010) Development of Macroporous Poly(ethylene glycol) Hydrogel arrays within microfluidic channels. Biomacromol. 11: 3316–3324.

    Article  CAS  Google Scholar 

  41. Dendukuri, D., S. S. Gu, D. C. Pregibon, T. A. Hatton, and P. S. Doyle (2007) Stop-flow lithography in a microfluidic device. Lab Chip 7: 818–828.

    Article  CAS  Google Scholar 

  42. Li, Z. and M. P. Deutscher (2008) Analyzing the decay of stable RNAs in E. coli. Methods Enzymol. 447: 31–45.

    Article  CAS  Google Scholar 

  43. Deutscher, M. P. (2003) Degradation of stable RNA in bacteria. J. Biol. Chem. 278: 45041–45044.

    Article  CAS  Google Scholar 

  44. Jung, S. and H. Yi (2015) Facile micromolding-based fabrication of biopolymeric–synthetic hydrogel microspheres with controlled structures for improved protein conjugation. Chem. Mater. DOI: 10.1021/acs.chemmater.5b00920.

    Google Scholar 

  45. Jung, S. and H. Yi (2014) An integrated approach for enhanced protein conjugation and capture with viral nanotemplates and hydrogel microparticle platforms via rapid bioorthogonal reactions. Langmuir 30: 7762–7770.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Tufts University, 4 Colby St., Medford, MA, 02155, USA

    Yader Duenas, JaeHun Lee, Sukwon Jung & Hyunmin Yi

Authors
  1. Yader Duenas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. JaeHun Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sukwon Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hyunmin Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hyunmin Yi.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Duenas, Y., Lee, J., Jung, S. et al. Multiplexed hydrogel microparticle suspension arrays for facile ribosomal RNA integrity assays. Biotechnol Bioproc E 20, 956–964 (2015). https://doi.org/10.1007/s12257-015-0265-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-015-0265-z

Keywords

Search

Navigation