Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis.

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 TGFß1 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.

Poly(N-(4-aminobutyl)-acrylamide) as mimetic polylysine for improving survival and differentiation of cerebellar granule neurons.

Poly(N-(4-aminobutyl)-acrylamide) (P4Am) and poly-d-lysine (PDL) possess the same butylamine side chain. The main difference is the peptide structure, which is in the main chain of PDL but in the side chain of P4Am. PDL has been extensively used in the preparation of neuronal cultures. We assumed neurons are sensitive enough to distinguish such structure difference so these two cationic polymers were compared at serial coating concentrations for culturing cerebellar granule neurons from 7-day-old Wistar rats in this study. Cellular viability and morphology assay showed no obvious difference for neurons cultured at high coating concentrations (>0.31 µg/mL) of these two polymers. In contrast, the difference in the peptide structure between P4Am and PDL could be distinguished by neurons at low coating concentrations (< 0.16 µg/mL). P4Am at low coating concentration could keep aggregates with three or four thick processes to support a more complete neural network with higher cellular viability than PDL. This suggests the interaction between neurons and the specific peptide structure of P4Am at low coating concentration was able to improve survival and differentiation of cultured cerebellar granule neurons. Although neural cells exhibited different morphologies and activities on high and low P4Am coating concentrations, immunofluorescence imaging confirmed most of cultured cells were functionally mature neurons, stained by neurofilament, synapsin I, and GAP43. The information should be useful for designing new biomaterials for regeneration of damaged circuits following disease or trauma. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1194-1201, 2018.