Biomedical Microdevices

, Volume 9, Issue 5, pp 729–736

Gene transcript amplification from cell lysates in continuous-flow microfluidic devices

  • Asensio Gonzalez
  • Doina Ciobanu
  • Michael Sayers
  • Noel Sirr
  • Tara Dalton
  • Mark Davies
Article

Abstract

Continuous-flow analysis, where samples circulate encapsulated in a carrier fluid is an attractive alternative to batch processing for high-throughput devices that use the polymerase chain reaction (PCR). Challenges of continuous-flow prototypes include the hydrodynamic and biological incompatibility of the carrier fluid, microchannel fouling, sample carryover and the integration of a nucleic acid extraction and reverse transcription step. We tested two homemade, continuous-flow thermocycler microdevices for amplification of reverse-transcribed messages from cell lysates without nucleic acid extraction. Amplification yield and specificity were assessed with state-of-the-art, real-time quantitative equipment. Carryover contamination between consecutive samples was absent. Amplification specificity and interference by genomic DNA were optimized by primer design. Robust detection of the low-copy transcript CLIC5 from 18 cells per microliter is demonstrated in cultured lymphoblasts. The results prove the concept that the development of micro-total analysis systems (μ-TAS) for continuous gene expression directly from cell suspensions is viable with current technology.

Keywords

PCR Gene expression Microfluidic devices Continuous-flow μ-TAS 

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Asensio Gonzalez
    • 2
  • Doina Ciobanu
    • 1
  • Michael Sayers
    • 1
  • Noel Sirr
    • 1
  • Tara Dalton
    • 1
  • Mark Davies
    • 1
  1. 1.Stokes Research InstituteUniversity of LimerickLimerickIreland
  2. 2.Northern Ireland Regional Histocompatibility and Immunogenetics Laboratory, Blood Transfusion ServiceBelfast City HospitalBelfastUK

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