Nanoliter high throughput quantitative PCR

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

Design, operation and applications of a visible-light confocal scanning Fourier transform Raman microscope for volumetric Raman spectrochemical imaging

A new type of confocal Raman microscope called a Fourier transform confocal Raman microscope (FT-CRM) was designed, built and characterized with respect to its spatio-spectral imaging properties. Several different applications of the FT-CRM are presented that take advantage of its unique spectral and spatial imaging characteristics. The instrument combines focused illumination with spatially-filtered detection in a confocal optical configuration to collect photons scattered from a diffraction-limited volume in the sample (typically ${<}5 times10 sp{-18} m sp3)$ and reject photons from outside that region. The molecular vibrational information encoded in the inelastic, or Raman, spectral component of light scattered from the confocal volume is measured with a visible light Fourier transform Raman spectrometer. By scanning the sample relative to the confocal volume, a volumetric Raman spectrochemical image of the sample can be constructed.Raman scattering is an inherently inefficient process; hence an optimal radius pinhole must be found that balances the FT-CRM optical throughput against the microscope spatial resolution and image contrast. Detailed experimental measurements mapped out the FT-CRM spatial response (axial and lateral), optical throughput and image signal-to-background and signal-to-noise ratios as a function of pinhole radius. Excellent agreement was found between these measurements and the predictions of a theoretical microscope model also developed as part of this thesis. Several applications of the FT-CRM included volumetric compositional imaging of three-dimensional chemically inhomogeneous materials such as cellulose and polyester fibers in water or two immiscible optically-similar liquids, water and trichloroehthylene, in a porous quartz sandstone matrix. The potential of the FT-CRM for non-invasive spectrochemical detection and imaging through a turbid tissue-like medium was demonstrated and a new spectral estimator, Fast Orthogonal Search, was evaluated to replace the discrete Fourier transform to improve the microscope performance

Microplastic detection and identification by Nile red staining : towards a semi-automated, cost- and time-effective technique

Microplastic pollution is an issue of concern due to the accumulation rates in the marine environment combined with the limited knowledge about their abundance, distribution and associated environmental impacts. However, surveying and monitoring microplastics in the environment can be time consuming and costly. The development of cost-and time-effective methods is imperative to overcome some of the current critical bottlenecks in microplastic detection and identification, and to advance microplastics research. Here, an innovative approach for microplastic analysis is presented that combines the advantages of high-throughput screening with those of automation. The proposed approach used Red Green Blue (RGB) data extracted from photos of Nile red-fluorescently stained microplastics (50-1200 mu m) to train and validate a 'Plastic Detection Model' (PDM) and a 'Polymer Identification Model' (PIM). These two supervised machine learning models predicted with high accuracy the plastic or natural origin of particles (95.8%), and the polymer types of the microplastics (88.1%). The applicability of the PDM and the PIM was demonstrated by successfully using the models to detect (92.7%) and identify (80%) plastic particles in spiked environmental samples that underwent laboratorial processing. The classification models represent a semi-automated, high throughput and reproducible method to characterize microplastics in a straightforward, cost-and time-effective yet reliable way

Nanoliter high-throughput RT-qPCR: a statistical analysis and assessment

Biomarkers discovered from gene expression profiles using hybridization microarrays have made great inroads in the diagnosis and development of safer and efficacious drugs. The candidate gene set is biologically validated by quantitative measurement with reverse transcriptase quantitative PCR (RT-qPCR) and is an effective strategy when implemented with microplates if the number of candidate genes and samples is small. With the trend toward informative candidate gene panels increasing from tens to hundreds of genes and sample cohorts exceeding several hundred, an alternative fluidic approach is needed that preserves the intrinsic analytical precision, large dynamic range, and high sensitivity of RT-qPCR, yet is scalable to high throughputs. We have evaluated the performance of a nanoliter fluidic system that enables up to 3072 nanoliter RT-qPCR assays simultaneously in a high-density array format. We measured the transcription from two different adult human tissues to assess measurement reproducibility across replicates, measurement accuracy, precision, specificity, and sensitivity; determined the false positive rate (FPR) and false negative rate (FNR) of the expressed transcript copies; and determined differences in kinase gene expression reflecting tissue and dosage differences. Using our methodology, we confirm the potential of this technology in advancing pharmaceutical research and development

High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer

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High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

International audienceMining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility