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Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection

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dc.title Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection en
dc.contributor.author Húska, Dalibor
dc.contributor.author Adam, Vojtěch
dc.contributor.author Hubálek, Jaromír
dc.contributor.author Trnková, Libuše
dc.contributor.author Horna, Aleš
dc.contributor.author Vajtr, David
dc.contributor.author Havel, Ladislav
dc.contributor.author Kizek, René
dc.relation.ispartof Talanta
dc.identifier.issn 0039-9140 OCLC, Ulrich, Sherpa/RoMEO, JCR
dc.date.issued 2009
utb.relation.volume 79
utb.relation.issue 2
dc.citation.spage 402
dc.citation.epage 411
dc.type article
dc.language.iso en
dc.publisher Elsevier Science B.V. en
dc.identifier.doi 10.1016/j.talanta.2009.04.007
dc.relation.uri https://www.sciencedirect.com/science/article/pii/S0039914009002902
dc.subject mRNA en
dc.subject Isolation en
dc.subject Magnetic particle and bead en
dc.subject Cyclic voltammetry en
dc.subject Square wave voltammetry en
dc.subject Brain tissue en
dc.subject Maize plants en
dc.subject Cadmium en
dc.description.abstract Easy, efficient and low demanding separation of mRNA from biological material is needed to study gene expression and to use in chip technologies. It is common knowledge that each mRNA molecule contains sequence of 25 adenines. This feature can be used for binding mRNA on the surface of the particles coated by thymine chains. The present work reports on suggesting and optimizing of mRNA separation and detection from biological material via paramagnetic microparticles coupled with electrochemical detection. Primarily we optimized cyclic and square wave voltammetric conditions to detect poly(A),which was used as standard to mimic behaviour of mRNA. Under the optimized square wave voltammetric conditions (frequency 280 Hz, accumulation time 200 s, supporting electrolyte and its temperature: acetate buffer 4.6 and 35 degrees C) we estimated detection limit down to 1 ng of poly(A) per ml. To enhance effectiveness and repeatability of isolation of nucleic acid automated approach for rinsing and hybridizing was proposed. We optimized the whole procedure and experimental conditions. Using automated way of isolation and under optimized conditions the yield of poly(A) (isolated concentration of poly(A)/given concentration of poly(A)*100) was approximately 75%. The suggested and optimized method for poly(A) isolation and detection was utilized for the analysis of brain tissues of patients with traumatic brain injury. The total amount of isolated mRNA varied from 40 to 760 g of mRNA per g of brain tissue. The isolation of mRNA from six samples per run was not longer than 2.5 h. Moreover, we applied the optimized procedure on fully automated pipetting instrument to isolate mRNA. The instrument was successfully tested on the analysis of extracts from roots of maize plants treated with cadmium(II) ions. (C) 2009 Elsevier B.V. All rights reserved. en
utb.faculty University Institute
dc.identifier.uri http://hdl.handle.net/10563/1001702
utb.identifier.rivid RIV/70883521:28610/09:63508006!RIV10-MSM-28610___
utb.identifier.obdid 43861800
utb.identifier.scopus 2-s2.0-67549135482
utb.identifier.wok 000268216900046
utb.source j-riv
utb.ou Centre of Polymer Systems
utb.contributor.internalauthor Horna, Aleš
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