Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips

Abstract

The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (microPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the microPCR chip down to 5 starting target molecules using TaqMan chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 microl assays. The high sensitivity and quantification capability of the microPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.

Inhibitory effect of common microfluidic materials on PCR outcome

“…Conventional tubes, multiwell plates, and microchips for PCR are fabricated from PP [49]. PTFE is a synthetic fluoropolymer, an inert material that has already been used in microfluidics PCR [18].…”

Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

“…Following the early development of the conventional close channel microfluidics based PCR micro-devices [6][7][8][9], handling the PCR sample volume in form of rapidly dispensed, discrete microliter or smaller droplets has become a preferred method of choice for PCR micro-devices [10,11]. Most microfluidic PCR systems have focused primarily on either reducing the PCR reaction volume (down to few hundred nanoliter) [12][13][14] or reducing the PCR reaction time (~10 min) [12][13][14][15][16]. The requirement of high surfactant concentration in the continuous oil phase to stabilize the PCR droplets, lack of individual addressing of the multiple droplets and the excessive need for off-chip overhead (pumps, plumbing, valves) are just a few of the challenging issues driving the development of miniaturized PCR set-ups towards droplet microfluidic (DMF).…”

“…On a miniaturized scale, RT-PCR reactions have been achieved by utilizing microfluidic methods for manipulating nucleic acid samples and PCR reagents including the use of continuous flow techniques [6][7][8][9] and discrete droplet based microfluidics [10,11]. The majority of microfluidic implementations for RT-PCR assays have been targeted to gene expression analysis, where a large amount of genomic molecules has helped towards lowering the reaction volumes to sub-microliters [12][13][14]. However, in clinical diagnostic applications, for the detection of trace quantities of viral RNA in a matrix sample that often contains an abundance of genomic DNA from the human host, the emphasis is placed on the reliable, rapid detection [15].…”

Kinetic characteristics of continuous flow polymerase chain reaction chip: A numerical investigation

“…There are three kinds of micro PCR chip [2]: One is chamber-shaped PCR chip [7,8], which works more like a traditional PCR instrument but with a shrunken reaction volume. Integrated heater and sensor will ramp the temperature inside the reaction chamber with a much faster speed than the traditional PCR instrument and thereby facilitate a rapid DNA amplification.…”