Nanoliter High Throughput Quantitative PCR

BioTrove Inc, 12 Gill Street, Suite 4000, Woburn, MA 01810, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2006; 34(18):e123. DOI: 10.1093/nar/gkl639
Source: PubMed


Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of
RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method
for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically
limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes
simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described
here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array
of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with
an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume,
a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

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    • "This is an open access article under the CC BY-NC-ND license ( 4.0/). measurement [11] [12]. This facilitates thousands of assays to be performed in parallel. "
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    ABSTRACT: PCR is a common and often indispensable technique used in medical and biological research labs for a variety of applications. Real-time quantitative PCR (RT-qPCR) has become a definitive technique for quantitating differences in gene expression levels between samples. Yet, in spite of this importance, reliable methods to quantitate nucleic acid amounts in a higher throughput remain elusive. In the following paper, a unique design to quantify gene expression levels at the nanoscale in a continuous flow system is presented. Fully automated, high-throughput, low volume amplification of deoxynucleotides (DNA) in a droplet based microfluidic system is described. Unlike some conventional qPCR instrumentation that use integrated fluidic circuits or plate arrays, the instrument performs qPCR in a continuous, micro-droplet flowing process with droplet generation, distinctive reagent mixing, thermal cycling and optical detection platforms all combined on one complete instrument. Detailed experimental profiling of reactions of less than 300 nl total volume is achieved using the platform demonstrating the dynamic range to be 4 order logs and consistent instrument sensitivity. Furthermore, reduced pipetting steps by as much as 90% and a unique degree of hands-free automation makes the analytical possibilities for this instrumentation far reaching. In conclusion, a discussion of the first demonstrations of this approach to perform novel, continuous high-throughput biological screens is presented. The results generated from the instrument, when compared with commercial instrumentation, demonstrate the instrument reliability and robustness to carry out further studies of clinical significance with added throughput and economic benefits.
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    • "These new technologies use only small volumes of samples and reagents in the reactions, and combine high accuracy with simplicity. A fewer number of steps, together with automation using pipetting stations, decreases both the time required for development of the experimental phase and the likely errors associated with manual pipetting, allowing several tests to be performed simultaneously at low cost [17]. "
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    • "OpenArray Ò Real-Time PCR System (Life technologies) based on nanoliter qPCR (Morrison et al. 2006;Dixon et al. 2009). The information generated by RNA-seq and microarrays is thus complementary, highly congruent and in most cases allows for an integrative analysis of cellular pathways. "
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