title: Gene transcript amplification from cell lysates in continuous-flow microfluidic devices.
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 (micro-TAS) for continuous gene expression directly from cell suspensions is viable with current technology.
authors.affiliation: Northern Ireland Regional Histocompatibility and Immunogenetics Laboratory, Blood Transfusion Service, Belfast City Hospital, Belfast, BT9 7TS, and Stokes Research Institute, University of Limerick, Ireland, UK. asensio.gonzalez@bll.n-i.nhs.uk
authors.forename: Asensio
authors.initials: A
authors.lastname: Gonzalez
authors.forename: Doina
authors.initials: D
authors.lastname: Ciobanu
authors.forename: Michael
authors.initials: M
authors.lastname: Sayers
authors.forename: Noel
authors.initials: N
authors.lastname: Sirr
authors.forename: Tara
authors.initials: T
authors.lastname: Dalton
authors.forename: Mark
authors.initials: M
authors.lastname: Davies
mesh_terms: D045744:Cell Line, Tumor; D018118:Chloride Channels / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004867:Equipment Design; D015870:Gene Expression; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology; D013696:Temperature; D014158:Transcription, Genetic
chemical_list: C411466:CLIC5 protein, human; D018118:Chloride Channels; D008840:Microfilament Proteins; D004247:DNA