Complete sequence-based pathway analysis by differential on-chip DNA and RNA extraction from a single cell

Strijp, Dianne van; Vulders, Roland C. M.; Larsen, Nicholas; Schira, Julien; Baerlocher, Loïc; Driel, M.A. Van; Pødenphant, Marie; Hansen, Torbjsrn; Mir, Kalim U.; Olesen, Tom; Verhaegh, Wim; Marie, Rodolphe; Zaag, P. J. van der

doi:10.1038/s41598-017-10704-4

Cited by 17 publications

(31 citation statements)

References 28 publications

(49 reference statements)

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“…1a and S1 †) is placed in the instrument allowing bright field and fluorescence imaging, the control of the device temperature and connection of the device inlets (cell, B1 and B2 inlet, Fig. 1b) to a multi-channel air pressure controller 7 ( Fig. S1g †).…”

Section: Resultsmentioning

confidence: 99%

“…Then alkaline lysis solution from the REPLI-g single cell kit was added to the outlet well. 7 2.4.3 Whole genome amplification in device outlets. Whole genome amplification (WGA) was performed using REPLI-g single cell or the Qiagen REPLI-g UltraFast Mini Kit (Qiagen) which are based on multiple displacement amplification (MDA) technology to amplify gDNA from small samples.…”

Section: On Chip Protocol 2: (13 Cells Processed In Eindhoven)mentioning

confidence: 99%

“…This lysis solution enables collecting the RNA prior to collecting the DNA of the trapped cell. 7 Alternatively, an alkaline lysis buffer (D2, pH above 12) provided with the Repli-g UltraFast kit was used in Oxford for the analysis of the 39 single cells from the LS174T and LS180 cell lines, and from two fresh tumour samples (Fig. S4 †).…”

Section: Cell Capture and Lysismentioning

confidence: 99%

“…In all the samples, the cell cytosol is removed by a solution of 0.5% Triton-X100 in 0.5× Tris borate EDTA (TBE) buffer (Sigma), containing 1 μM of YOYO-1 dye (ThermoFisher Scientific). 7 The DNA was extracted from the cells by proteolysis buffer containing >200 μg mL −1 proteinase K (Qiagen), 0.5% v/v Triton-X100 in 0.5× TBE buffer. Then alkaline lysis solution from the REPLI-g single cell kit was added to the outlet well.…”

Section: On Chip Protocol 2: (13 Cells Processed In Eindhoven)mentioning

confidence: 99%

“…3,4 There has therefore been increasing focus on the development of methods that obtain molecular information from single cells by isolating and sequencing their DNA and mRNA content. [5][6][7] Similarly, in metagenomics, where bacteria, fungi and other microbes may not be culturable, a robust single cell analysis is important to evaluate the genomes of the distinct microbes present in a sample. 8 In addition to untangling heterogeneity, the single cell methods are relevant to cases when only a small number of cells is available, for example in the analysis of circulating tumour cells 9,10 and circulating fetal cells in maternal blood.…”

Section: Introductionmentioning

confidence: 99%

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Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

et al. 2018

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Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

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Section: Resultsmentioning

confidence: 99%

Section: On Chip Protocol 2: (13 Cells Processed In Eindhoven)mentioning

confidence: 99%

Section: Cell Capture and Lysismentioning

confidence: 99%

Section: On Chip Protocol 2: (13 Cells Processed In Eindhoven)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

et al. 2018

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Implementing microwell slides for detection and isolation of single circulating tumor cells from complex cell suspensions

et al. 2022

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Cell loss during detection and isolation of circulating tumor cells (CTCs) is a challenge especially when label‐free pre‐enrichment technologies are used without the aid of magnetic particles. Although microfluidic systems can remove the majority of “contaminating” white blood cells (WBCs), their remaining numbers are still impeding single CTC isolation, thus making additional separation steps needed. This study aimed to develop a workflow from blood‐to‐single CTC for complex cell suspensions by testing two microwell formats. In the first step, different cell lines were used to compare the performances of Sievewell™ 370 K (TOK, Japan) and CellCelector™ Nanowell U25 (ALS Automated Lab Solutions, Germany) slides for cell labelling and single‐cell micromanipulation. Confounding levels of auto‐fluorescence inherent to different plastic materials used to cast the microwells, staining recovery rates, and cell isolation rates were determined. In the second step, three different blood preservation tubes were tested for RNA analysis. Lastly, the established workflow was applied to isolate CTCs from peripheral blood samples obtained from metastasized breast cancer (mBC) patients for single‐cell DNA and RNA analysis. The detection of CTCs in Sievewell slides profit from better signal‐to‐noise ratios in the fluorescence channels mainly used for CTC detection. In addition, due to its design, Sievewell supports direct in situ CTC labelling, which minimizes cell loss and leads to single‐cell recovery rates after staining of approx. 94%. Detection of PIK3CA mutations in single CTCs verified the applicability of the workflow for the analysis of genomic DNA of CTCs. Furthermore, combined with blood preservation up to 48 h at room temperature in LBguard tubes, panel RT‐PCR transcript analysis was successful for single cell line cells and CTCs, respectively. The combined use of Sievewell microwell slides and CellCelector™ automated micromanipulation system improves single CTC detection, labelling and isolation from complex cell suspensions. This approach is especially valuable when samples of high cellular content are processed.

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Beyond single cells: microfluidics empowering multiomics analysis

Tian,

Lin,

Yang

2023

Anal Bioanal Chem

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Complete sequence-based pathway analysis by differential on-chip DNA and RNA extraction from a single cell

Cited by 17 publications

References 28 publications

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

Implementing microwell slides for detection and isolation of single circulating tumor cells from complex cell suspensions

Beyond single cells: microfluidics empowering multiomics analysis

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