Rapid, quantitative, reverse transcription PCR in a polymer microfluidicchip

Saunders, D. Curtis; Holst, Gregory L.; Phaneuf, Christopher R.; Pak, Nikita; Marchese, Matthew; Sondej, Nicholas; McKinnon, Michael; Forest, Craig R.

doi:10.1016/j.bios.2013.01.019

Cited by 26 publications

(9 citation statements)

References 48 publications

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“…To achieve imaging compatibility, one can adopt either a careful design for the geometry, or alternative transparent materials such as ITO (indium tin oxide)-coated glass. Infrared laser has also been used to efficiently heat up aqueous solutions in a microfluidic chamber by selecting radiation wavelengths to match absorption spectra of a particular liquid/solution [67,68]. This method features rapid temperature ramping ability, while meeting the requirements for laser instrumentation.…”

Section: Modeling the Physical And Chemical Environmentmentioning

confidence: 99%

Microfluidic and mathematical modeling of aquatic microbial communities

Liu

Giometto

2020

Anal Bioanal Chem

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Aquatic microbial communities contribute fundamentally to biogeochemical transformations in natural ecosystems, and disruption of these communities can lead to ecological disasters such as harmful algal blooms. Microbial communities are highly dynamic, and their composition and function are tightly controlled by the biophysical (e.g., light, fluid flow, and temperature) and biochemical (e.g., chemical gradients and cell concentration) parameters of the surrounding environment. Due to the large number of environmental factors involved, a systematic understanding of the microbial community-environment interactions is lacking. In this article, we show that microfluidic platforms present a unique opportunity to recreate well-defined environmental factors in a laboratory setting in a high throughput way, enabling quantitative studies of microbial communities that are amenable to theoretical modeling. The focus of this article is on aquatic microbial communities, but the microfluidic and mathematical models discussed here can be readily applied to investigate other microbiomes.

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Section: Modeling the Physical And Chemical Environmentmentioning

confidence: 99%

Microfluidic and mathematical modeling of aquatic microbial communities

Liu

Giometto

2020

Anal Bioanal Chem

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“…In a glass flow-through chip, Yu et al used a 50 W tungsten lamp to cycle a 10 μL sample, realizing 40 cycles in 35 min with ~2–6°C/s heating and ~1.5–2.0°C/s cooling [ 69 ]. With a 700 mW IR laser, water-cooled fixture, and polymer flow-through chip, Saunders et al heated (3.3°C/s) and cooled (3.9°C/s) 1 μL samples to accomplish qRT-PCR in about 1 hour [ 125 ].…”

Section: Background: Thermal Cycling Technologiesmentioning

confidence: 99%

The Rotary Zone Thermal Cycler: A Low-Power System Enabling Automated Rapid PCR

et al. 2015

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Advances in molecular biology, microfluidics, and laboratory automation continue to expand the accessibility and applicability of these methods beyond the confines of conventional, centralized laboratory facilities and into point of use roles in clinical, military, forensic, and field-deployed applications. As a result, there is a growing need to adapt the unit operations of molecular biology (e.g., aliquoting, centrifuging, mixing, and thermal cycling) to compact, portable, low-power, and automation-ready formats. Here we present one such adaptation, the rotary zone thermal cycler (RZTC), a novel wheel-based device capable of cycling up to four different fixed-temperature blocks into contact with a stationary 4-microliter capillary-bound sample to realize 1-3 second transitions with steady state heater power of less than 10 W. We demonstrate the utility of the RZTC for DNA amplification as part of a highly integrated rotary zone PCR (rzPCR) system that uses low-volume valves and syringe-based fluid handling to automate sample loading and unloading, thermal cycling, and between-run cleaning functionalities in a compact, modular form factor. In addition to characterizing the performance of the RZTC and the efficacy of different online cleaning protocols, we present preliminary results for rapid single-plex PCR, multiplex short tandem repeat (STR) amplification, and second strand cDNA synthesis.

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“…In comparison, microfluidic quantitative reverse transcription polymerase chain reaction (RT-qPCR), which detects gene expression through the creation of complementary DNA (cDNA) transcripts from RNA offers large dynamic ranges as well as high sensitivity and accuracy 27, 28 . For example, a microfluidic device for gene expression measurements was developed employing an open-loop infrared laser-based thermal control system where RNA templates from the lysate of cells can be quantitatively analyzed 29 . A microchip has also been presented to capture single cells and reverse transcribe messenger RNA (mRNA) in cell lysate to cDNA, which is fed into a commercial system (BioMark, Fluidigm) for analysis 30 .…”

Section: Introductionmentioning

confidence: 99%

A bead-based microfluidic approach to integrated single-cell gene expression analysis by quantitative RT-PCR

et al. 2015

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Gene expression analysis at the single-cell level is critical to understanding variations among cells in heterogeneous populations. Microfluidic reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is well suited to gene expression assays of single cells. We present a microfluidic approach that integrates all functional steps for RT-qPCR of a single cell, including isolation and lysis of the cell, as well as purification, reverse transcription and quantitative real-time PCR of messenger RNA in the cell lysate. In this approach, all reactions in the multi-step assay of a single lysed cell can be completed on microbeads, thereby simplifying the design, fabrication and operation of the microfluidic device, as well as facilitating the minimization of sample loss or contamination. In the microfluidic device, a single cell is isolated and lysed; mRNA in the cell lysate is then analyzed by RT-qPCR using primers immobilized on microbeads in a single microchamber whose temperature is controlled in closed loop via an integrated heater and temperature sensor. The utility of the approach was demonstrated by the analysis of the effects of the drug (methyl methanesulfonate, MMS) on the induction of the cyclin-dependent kinase inhibitor 1a (CDKN1A) in single human cancer cells (MCF-7), demonstrating the potential of our approach for efficient, integrated single-cell RT-qPCR for gene expression analysis.

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Rapid, quantitative, reverse transcription PCR in a polymer microfluidicchip

Cited by 26 publications

References 48 publications

Microfluidic and mathematical modeling of aquatic microbial communities

Microfluidic and mathematical modeling of aquatic microbial communities

The Rotary Zone Thermal Cycler: A Low-Power System Enabling Automated Rapid PCR

A bead-based microfluidic approach to integrated single-cell gene expression analysis by quantitative RT-PCR

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