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 ...Continue Reading
References
Nov 15, 2000·FEMS Microbiology Reviews·J Fröhlich, H König
Oct 23, 2003·Analytical Chemistry·Aaron R WheelerAntoine Daridon
Oct 6, 2005·Genome Research·Martin BengtssonMikael Kubista
Apr 29, 2006·Analytical Chemistry·Joshua S MarcusStephen R Quake
Nov 14, 2006·Proceedings of the National Academy of Sciences of the United States of America·Luigi WarrenStephen R Quake
Sep 26, 2007·Expert Review of Molecular Diagnostics·Virginia EspinaLance A Liotta
Aug 7, 2008·PloS One·Simant DubeRamesh Ramakrishnan
Jan 6, 2009·Nature Methods·Alison M SkelleyJoel Voldman
Jan 13, 2010·Methods : a Companion to Methods in Enzymology·Anders Ståhlberg, Martin Bengtsson
Mar 31, 2011·Analytical Chemistry·Rebecca SandersCarole A Foy
Jun 18, 2011·Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology·Jan TheysMarc Vooijs
Oct 11, 2011·Journal of Virological Methods·Richard Allen WhiteKenneth Curr
Nov 28, 2012·Diagnostic Microbiology and Infectious Disease·Ruth Hall Sedlak, Keith R Jerome
Apr 16, 2013·Trends in Biotechnology·Kerry Routenberg LoveJ Christopher Love
Jun 20, 2013·Theranostics·Bin Hong, Youli Zu
Jul 31, 2013·Nature Reviews. Genetics·Ehud ShapiroSten Linnarsson
Sep 3, 2013·Nature Methods·Christopher M HindsonMuneesh Tewari
Sep 5, 2013·Clinical Chemistry·Tanis C DingleKeith R Jerome
Sep 16, 2014·International Journal of Cancer. Journal International Du Cancer·Jacques FerlayFreddie Bray
Jan 23, 2015·Scientific Reports·Georg WelzelStefan Schuster
Jan 30, 2015·Plant Methods·Nejc RačkiMaja Ravnikar
Nov 9, 2016·Frontiers in Cell and Developmental Biology·Ping HuGlenn Deng
Jan 6, 2017·CA: a Cancer Journal for Clinicians·Rebecca L SiegelAhmedin Jemal
Jul 14, 2017·Biomolecular Detection and Quantification·Amin ForootanMikael Kubista