Rapid, quantitative, reverse transcription PCR in a polymer microfluidic chip.
Biosens Bioelectron
; 44: 222-8, 2013 Jun 15.
Article
em En
| MEDLINE
| ID: mdl-23434757
ABSTRACT
Quantitative PCR (qPCR) techniques have become invaluable, high-throughput tools to study gene expression. However, the need to measure gene expression patterns quickly and affordably, useful for applications such as stem cell biomanufacturing requiring real-time observation and control, has not been adequately met by rapid qPCR instrumentation to date. We report a reverse transcription, microfluidic qPCR system and its application to DNA and RNA amplification measurement. In the system, an environmental control fixture provides mechanical and thermal repeatability for an infrared laser to achieve both accurate and precise open-loop temperature control of 1 µl reaction volumes in a low-cost polymer microfluidic chip with concurrent fluorescence imaging. We have used this system to amplify serial dilutions of λ-phage DNA (10(5)-10(7) starting copies) and RNA transcripts from the GAPDH housekeeping gene (5.45 ng total mouse embryonic stem cell RNA) and measured associated standard curves, efficiency (57%), repeatability (~1 cycle threshold), melting curves, and specificity. This microfluidic qRT-PCR system offers a practical approach to rapid analysis (~1 h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easy setup.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Viral
/
RNA
/
Bacteriófago lambda
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Reação em Cadeia da Polimerase Via Transcriptase Reversa
/
Técnicas Analíticas Microfluídicas
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article