Microfluidic extraction and digital quantification of circulating cell-free DNA from serum

International audienc

Facile Production of Large-Area Cell Arrays Using Surface-Assembled Microdroplets

Techniques that enable the spatial arrangement of living cells into defined patterns are broadly applicable to tissue engineering, drug screening, and cell–cell investigations. Achieving large-scale patterning with single-cell resolution while minimizing cell stress/damage is, however, technically challenging using existing methods. Here, a facile and highly scalable technique for the rational design of reconfigurable arrays of cells is reported. Specifically, microdroplets of cell suspensions are assembled using stretchable surface-chemical patterns which, following incubation, yield ordered arrays of cells. The microdroplets are generated using a microfluidic- based aerosol spray nozzle that enables control of the volume/size of the droplets delivered to the surface. Assembly of the cell-loaded microdroplets is achieved via mechanically induced coalescence using substrates with engineered surface-wettability patterns based on extracellular matrices. Robust cell proliferation inside the patterned areas is demonstrated using standard culture techniques. By combining the scalability of aerosol-based delivery and microdroplet surface assembly with user-defined chemical patterns of controlled functionality, the technique reported here provides an innovative methodology for the scalable generation of large-area cell arrays with flexible geometries and tunable resolution

PCR digitale en micro-compartiments

International audienc

Digital PCR compartmentalization I. Single-molecule detection of rare mutations.

Polymerase chain reaction based techniques have been widely used in laboratory settings. Several applications in oncology, virology or prenatal diagnosis require highly sensitive detection methods, which cannot be achieved with conventional techniques. Digital PCR (dPCR) was developed from the association of PCR and limiting dilution procedures. It is based on the compartmentalization of DNA molecules in small volumes. Controlling the size and the content of each compartment is crucial to obtain a high sensitivity with a single molecule resolution. Microfluidics offers promising tools to isolate DNA fragments such as microdroplets, microchambers or microwells with volumes ranging from few picoliters to nanoliters. The review provides an overview of recent developments of microfluidics dPCR platforms and how this technology can influence the management of cancer patients

The Microbiome Meets Nanotechnology: Opportunities and Challenges in Developing New Diagnostic Devices

International audienceMonitoring of the human microbiome is an emerging area of diagnostics for personalized medicine. Here, the potential of different nanomaterials and nanobiosensing technologies is reviewed for the development of novel diagnostic devices for the detection and measurement of microbiome-related biomarkers. Moreover, the current and future landscape of microbiome-based diagnostics is defined by exploring the advantages and disadvantages of current nanotechnology-based approaches, especially in the context of developing point-of-care (PoC) devices that would meet the international guidelines known as REASSURED (Real-time connectivity; Ease of specimen collection; Affordability; Sensitivity; Specificity; User-friendliness; Rapid & robust operation; Equipment-free; and Deliverability). Finally, the strategies of the latest international scientific consortia working in this field are analyzed, the current microbiome diagnostics market are reported and the principal ethical, legal, and societal issues related to microbiome R&D and innovation are discussed

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients

On-Chip Magnetic Extraction of Circulating Cell-Free DNA from Biological Samples

International audienceIn recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfDNA from peripheral blood has remained a challenge due to the low abundance and high fragmentation of these molecules. Here, we present a dynamic Magnetic ExTRactiOn (METRO) protocol using microfluidic fluidized bed technology to isolate circulating cfDNA from raw biological materials such as undiluted serum. This protocol maximizes the surface area for DNA binding within the chip in order to capture short DNA fragments. It uses only a few μL of sample and reagents. The protocol can be automated, and it is fully compatible with sensitive DNA amplification methods such as droplet-based digital PCR (ddPCR)

BRCA1 and RAD51C promotor methylation in human resectable pancreatic adenocarcinoma

International audienceBackground: Homozygous Recombination Deficiency (HRD) is associated with sensitivity to PARP-inhibitors (PARPi) in different cancer types. In pancreatic adenocarcinoma (PA) the main cause of HRD is BRCA1/2 germline mutation and patients with mutations in BRCA1/2 may benefit from PARPi. Recently other mechanisms leading to HRD were described in different cancer types, including gene mutations and epigenetic changes such as promoter hypermethylation. In PA, BRCA1 promoter hypermethylation, a known mechanism of gene silencing, was recently described. However, results are discordant between North American studies (0.7% of PA) and Asian ones (up to 60% of PA) and the association with HRD is not clear.Methods: Here, we developed 2 quantifications methods to explore BRCA1 and RAD51C promoter methylation in a series of 121 Formalin Fixed-Paraffin-Embedded (FFPE) specimens from resected PA without neoadjuvant treatment. The methylation-specific PCR was done with 2 different methods after DNA bisulfite conversion: a digital droplet PCR, and a PCR followed by capillary electrophoresis, to score the methylated / non methylated ratios in tumor samples. Methods were validated for specificity and sensibility using 100, 20, 10, 5 and 0% methylated commercial DNA for fragment analysis with a detection cutoff of 5-10%. Limit of blank was defined as 5 dropplets/20µL for RAD51C and 1 dropplet/20µL for BRCA1 for ddPCR. Samples were reviewed by a pathologist, macrodissected before DNA extraction to obtain 50-60% of tumoral cells. DNAs were treated for bisulfite conversion and analyzed using both methods in parallel to known positive and negative controls in each run.Results and conclusion: No methylation at BRCA1 or RAD51C was found in this series of PA suggesting that HRD gene promoter methylation is a rare event in European patients

Lysimachia tanakae Maxim.

原著和名: ミヤマコナスビ科名: サクラソウ科 = Primulaceae採集地: 奈良県 玉置山 (大和 玉置山)採集日: 1983/6/26採集者: 萩庭丈壽整理番号: JH036314国立科学博物館整理番号: TNS-VS-98631

False-positive evaluation in negative controls (human wild-type genomic DNA).

<p>In order to assess the false-positive (FP) events detected in negative control samples, we analysed by dPCR a collection of human wild-type only samples (genomic DNA, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159094#pone.0159094.s001" target="_blank">S1 Fig</a> for details) with the EGFR, KRAS and TP53 assays described. We used two different amounts of DNA input (20 and 60 ng, depicted by circles and squares respectively). The scatter plot displays the low dynamic range detection of three castPCR™ probes (EGFR p.T790M, TP53 p.R273H and p.R213*), where the number of FP events increased when using 60 ng of starting DNA material (lines represent the mean for each assay). At right, the table shows the LOB and LOD estimation of all assays (refer to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159094#pone.0159094.ref033" target="_blank">33</a>] for precise <i>formula</i>), calculated from the λ_FP of each test (where λ_FP is given by the mean number of false-positives obtained in all experiments realized with 20 ng input DNA). Mean value and standard deviation for each FP measurement for the different assays are shown, both for WT and MUT-DNA containing droplets. <i>(C)</i>, <i>castPCR™ probes; (T)</i>, <i>TaqMan® probes; (Z)</i>, <i>ZEN™ probes; N°</i>, <i>number; FP</i>, <i>false-positive; LOB</i>, <i>Limit of Blank; LOD</i>, <i>Limit of Detection; N/A</i>, <i>not applicable</i>.</p