Abstract
In a single-cell study, isolating and identifying single cells are essential, but these processes often require a large investment of time or money. The aim of this study was to isolate and analyse single cells using a novel platform, the PanelChip™ Analysis System, which includes 2500 microwells chip and a digital real-time polymerase chain reaction (dqPCR) assay, in comparison with a standard PCR (qPCR) assay. Through the serial dilution of a known concentration standard, namely pUC19, the accuracy and sensitivity levels of two methodologies were compared. The two systems were tested on the basis of expression levels of the genetic markers vimentin, E-cadherin, N-cadherin and GAPDH in A549 lung carcinoma cells at two known concentrations. Furthermore, the influence of a known PCR inhibitor commonly found in blood samples, heparin, was evaluated in both methodologies. Finally, mathematical models were proposed and separation method of single cells was verified; moreover, gene expression levels during epithelial–mesenchymal transition in single cells under TGFβ1 treatment were measured. The drawn conclusion is that dqPCR performed using PanelChip™ is superior to the standard qPCR in terms of sensitivity, precision, and heparin tolerance. The dqPCR assay is a potential tool for clinical diagnosis and single-cell applications.
Highlights
Lung cancer is the most common cause of cancer-related death in the world[1]
Four new methods of note have been developed: fluorescence-activated cell sorting[7], in which suspended cells are sorted at high speed by detecting droplets containing single cells that have been labelled with a fluorescent signal and light scatter characteristics; magnetic-activated cell sorting[8], in which the suspended cells are labelled with antibody-conjugated magnetic beads, sorted in a magnetic field; laser capture microdissection[9], in which a laser is used to melt the thermoplastic film, allowing individual cells to adhere to the melted film to obtain the cells; and microfluidics[10,11,12,13], in which physical, chemical, and biochemical parameters are combined to allow precise control of fluid-flow behaviour in a sub-millimetre-scale space to achieve cell separation
From 3.4 to 3.4 × 108 copies/μL of the initial plasmid DNA template, the Cq values obtained from both dqPCR and qPCR were linear in relation to the initial concentration of pUC19 plasmid DNA
Summary
Lung cancer is the most common cause of cancer-related death in the world[1]. Most of these deaths are caused by the metastasis of lung cancer tumors, leading to circulating tumor cells (CTCs) spreading cancer to other areas of the body[2,3,4]. It entails first using a microscope to observe specific cells, using micropipettes to aspirate the cells, and moving the cells to a collection vessel This technique allows direct access to a single cell, but the process is time-consuming and inefficient, meaning it is unable to achieve high throughput. Four new methods of note have been developed: fluorescence-activated cell sorting[7], in which suspended cells are sorted at high speed by detecting droplets containing single cells that have been labelled with a fluorescent signal and light scatter characteristics; magnetic-activated cell sorting[8], in which the suspended cells are labelled with antibody-conjugated magnetic beads, sorted in a magnetic field; laser capture microdissection[9], in which a laser is used to melt the thermoplastic film, allowing individual cells to adhere to the melted film to obtain the cells; and microfluidics[10,11,12,13], in which physical, chemical, and biochemical parameters are combined to allow precise control of fluid-flow behaviour in a sub-millimetre-scale space to achieve cell separation. RNA-seq libraries are subsequently prepared and sequenced using a sequencing system[16]
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