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High-throughput microfluidic single-cell RT-qPCR.

ABSTRACT: A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells. Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity. We apply this technology to 3,300 single-cell measurements of (i) miRNA expression in K562 cells, (ii) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and (iii) single nucleotide variant detection in primary lobular breast cancer cells. The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed.

SUBMITTER: White AK

PROVIDER: S-EPMC3161570 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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High-throughput microfluidic single-cell RT-qPCR.

White Adam K AK VanInsberghe Michael M Petriv Oleh I OI Hamidi Mani M Sikorski Darek D Marra Marco A MA Piret James J Aparicio Samuel S Hansen Carl L CL

Proceedings of the National Academy of Sciences of the United States of America 20110801 34

A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells. Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. In addition to higher throughput an ...[more]

PMID: 21808033

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Project description:To incorporate genomics data into environmental assessments a mechanistic perspective of interactions between chemicals and induced biological processes needs to be developed. Since chemical compounds with structural similarity often induce comparable biological responses in exposed animals, gene expression signatures can serve as a starting point for the assessment of chemicals and their toxicity, but only when relevant and stable gene panels are available. To design such a panel, we isolated differentially expressed gene fragments from the soil arthropod Folsomia candida, a species often used for ecotoxicological testing. Animals were exposed to two chemically distinct compounds, being a metal (cadmium) and a polycyclic aromatic hydrocarbon (phenanthrene). We investigated the affected molecular responses resulting from either treatment and developed and validated 44 qPCR assays for their responses using a high throughput nano-liter RT-qPCR platform for the analysis of the samples.Suppressive subtractive hybridization (SSH) was used to retrieve stress-related gene fragments. SSH libraries revealed pathways involved in mitochondrial dysfunction and protein degradation for cadmium and biotransformation for phenanthrene to be overrepresented. Amongst a small cluster of SSH-derived cadmium responsive markers were an inflammatory response protein and an endo-glucanase. Conversely, cytochrome P450 family 6 or 9 was specifically induced by phenanthrene. Differential expressions of these candidate biomarkers were also highly significant in the independently generated test sample set. Toxicity levels in different training samples were not reflected by any of the markers' intensity of expressions. Though, a model based on partial least squares differential analysis (PLS-DA) (with RMSEPs between 9 and 22% and R(2)s between 0.82 and 0.97) using gene expressions of 25 important qPCR assays correctly predicted the nature of exposures of test samples.For the application of molecular bio-indication in environmental assessments, multivariate analyses obviously have an added value over univariate methods. Our results suggest that compound discrimination can be achieved by PLS-DA, based on a hard classification of the within-class rankings of samples from a test set. This study clearly shows that the use of high throughput RT-qPCR could be a valuable tool in ecotoxicology combining high throughput with analytical sensitivity.

Project description:BackgroundNormalization is critical for accurate gene expression analysis. A significant challenge in the quantitation of gene expression from biofluids samples is the inability to quantify RNA concentration prior to analysis, underscoring the need for robust normalization tools for this sample type. In this investigation, we evaluated various methods of normalization to determine the optimal approach for quantifying microRNA (miRNA) expression from biofluids and tissue samples when using the TaqMan® Megaplex™ high-throughput RT-qPCR platform with low RNA inputs.FindingsWe compared seven normalization methods in the analysis of variation of miRNA expression from biofluid and tissue samples. We developed a novel variant of the common mean-centering normalization strategy, herein referred to as mean-centering restricted (MCR) normalization, which is adapted to the TaqMan Megaplex RT-qPCR platform, but is likely applicable to other high-throughput RT-qPCR-based platforms. Our results indicate that MCR normalization performs comparable to or better than both standard mean-centering and other normalization methods. We also propose an extension of this method to be used when migrating biomarker signatures from Megaplex to singleplex RT-qPCR platforms, based on the identification of a small number of normalizer miRNAs that closely track the mean of expressed miRNAs.ConclusionsWe developed the MCR method for normalizing miRNA expression from biofluids samples when using the TaqMan Megaplex RT-qPCR platform. Our results suggest that normalization based on the mean of all fully observed (fully detected) miRNAs minimizes technical variance in normalized expression values, and that a small number of normalizer miRNAs can be selected when migrating from Megaplex to singleplex assays. In our study, we find that normalization methods that focus on a restricted set of miRNAs tend to perform better than methods that focus on all miRNAs, including those with non-determined (missing) values. This methodology will likely be most relevant for studies in which a significant number of miRNAs are not detected.

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High-throughput microfluidic single-cell RT-qPCR.

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High-throughput microfluidic single-cell RT-qPCR.

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