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.
    06/2015; 4. DOI:10.1016/j.bdq.2015.04.003
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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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    ABSTRACT: Hearing loss is the most common sensory deficit in humans, affecting approximately 10% of the global population. In developed countries, one in every 500 individuals suffers from severe to profound bilateral sensorineural hearing loss. For those up to 5 years old, the proportion is higher, at 2.7 in 1000 individuals, and for adolescents the average is 3.5 in 1000. Among the causes of hearing loss, more than 50% are related to genetic factors. To date, nearly 150 loci and 64 genes have been associated with hearing loss. Mutations in the GJB2 gene, which encodes connexin 26, constitute the main genetic cause. So far, more than 300 variations have been described in this gene.As a response to the clinical and genetic heterogeneity of hearing loss and the importance of correct molecular diagnosis of individuals with hereditary hearing loss, this study worked in the optimization for a diagnostic protocol employing a high-throughput genotyping technology. For this work, was used the TaqMan(R) OpenArrayTM Genotyping platform (BioTrove Inc., Woburn, MA, USA). This is a high performance, high-throughput technology based on real-time PCR, which enables the evaluation of up to 3072 SNPs (Single Nucleotide Polymorphisms), point mutations, small deletions, and insertions, using a single genotyping plate. For the study, were selected the layout allowing to analyze 32 alterations in 96 individuals simultaneously. In the end, the generated results were validated by conventional techniques, as direct sequencing, Multiplex PCR and RFLP-PCR. A total of 376 individuals were analyzed, of which 94 were healthy controls, totaling 4 plates in duplicate. All 31 of the changes analyzed were present in the nuclear genes GJB2, GJB6, CRYL1, TMC1, SLC26A4, miR-96, and OTOF, and in the mitochondrial genes MT-RNR1 and MT-TS1. The reactions were subsequently validated by established techniques (direct sequencing, multiplex PCR, and RFLP-PCR) that had previously been used to perform molecular screening of hearing loss at the Human Genetics Laboratory of the Center for Molecular Biology and Genetic Engineering (CBMEG), at the State University of Campinas (UNICAMP). In total, 11,656 genotyping reactions were performed. Of these, only 351 reactions failed, representing approximately 3.01% of the total. The average accuracy of genotyping using the OpenArrayTM plates was 96.99%. The results demonstrated the accuracy, low cost, and good reproducibility of the technique, indicating that the TaqMan(R) OpenArrayTM Genotyping Platform is a useful and reliable tool for application in molecular diagnostic testing of hearing loss.
    BMC Medical Genetics 10/2013; 14(1):112. DOI:10.1186/1471-2350-14-112 · 2.08 Impact Factor
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    • "The high performance of RNA-seq to produce sequences without previous genomic information makes these techniques the most powerful approaches for gene discovery and de novo characterization of a whole transcriptome. In this way, the information generated by RNA-seq can be used for the development of custom microarrays for systematic transcriptome profiling (Wong et al. 2011), and other high-throughput tools for quantitative real-time PCR (qPCR), such as the 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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    ABSTRACT: Fish aquaculture is considered to be one of the most sustainable sources of protein for humans. Many different species are cultured worldwide, but among them, marine flatfishes comprise a group of teleosts of high commercial interest because of their highly prized white flesh. However, the aquaculture of these fishes is seriously hampered by the scarce knowledge on their biology. In recent years, various experimental 'omics' approaches have been applied to farmed flatfishes to increment the genomic resources available. These tools are beginning to identify genetic markers associated with traits of commercial interest, and to unravel the molecular basis of different physiological processes. This article summarizes recent advances in flatfish genomics research in Europe. We focus on the new generation sequencing technologies, which can produce a massive amount of DNA sequencing data, and discuss their potentials and applications for de novo genome sequencing and transcriptome analysis. The relevance of these methods in nutrigenomics and foodomics approaches for the production of healthy animals, as well as high quality and safety products for the consumer, is also briefly discussed.
    Genes & Nutrition 08/2012; 8(1). DOI:10.1007/s12263-012-0312-8 · 2.79 Impact Factor
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