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

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作者：

A Gonzalez，D Ciobanu，M Sayers，N Sirr，T Dalton，M Davies

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摘要：

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.

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关键词：

PCR Gene expression Microfluidic devices Continuous flow &#956 TAS

DOI：

10.1007/s10544-007-9083-1

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年份：

2007