Automated nucleic acids isolation using paramagnetic
microparticles coupled with electrochemical detection

J 2009

Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection

HÚSKA, Dalibor; Jaromír HUBÁLEK; Vojtěch ADAM; David VAJTR; Aleš HORNA et al.

Základní údaje

Originální název

Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection

Název česky

Automatická izolace nukleových kyselin s použitím paramagnetických mikročástic spojená s elektrochemickou detekcí

Autoři

HÚSKA, Dalibor; Jaromír HUBÁLEK; Vojtěch ADAM; David VAJTR; Aleš HORNA; Libuše TRNKOVÁ; Ladislav HAVEL a René KIZEK

Vydání

Talanta, Elsevier, 2009, 0039-9140

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10405 Electrochemistry

Stát vydavatele

Nizozemské království

Utajení

není předmětem státního či obchodního tajemství

Impakt faktor

Impact factor: 3.290

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14310/09:00029663

Organizační jednotka

Přírodovědecká fakulta

UT WoS

000268216900046

Klíčová slova anglicky

mRNA; Isolation; Magnetic particle and bead; Cyclic voltammetry; Square wave voltammetry; Brain tissue; Maize plants; Cadmium

Změněno: 8. 12. 2009 11:21, RNDr. Kamila Neplechová, Ph.D.

Anotace

ORIG CZ

V originále

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.

Česky

Práce se zabývá navržením a optimalizací metody separace a detekce mRNA z biologických materiálů pomocí paramagnetických mikročástic spojenou s elektrochemickou detekcí.

Návaznosti

GA102/08/1546, projekt VaV

Název: Miniaturizované inteligentní systémy a nanostrukturované elektrody pro chemické, biologické a farmaceutické aplikace (NANIMEL)

Citovat

HÚSKA, Dalibor; Jaromír HUBÁLEK; Vojtěch ADAM; David VAJTR; Aleš HORNA; Libuše TRNKOVÁ; Ladislav HAVEL a René KIZEK. Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection. Talanta. Elsevier, 2009, roč. 79, č. 2, 11 s. ISSN 0039-9140.

@article{858780,
   author = {Húska, Dalibor and Hubálek, Jaromír and Adam, Vojtěch and Vajtr, David and Horna, Aleš and Trnková, Libuše and Havel, Ladislav and Kizek, René},
   article_number = {2},
   keywords = {mRNA; Isolation; Magnetic particle and bead; Cyclic voltammetry; Square wave voltammetry; Brain tissue; Maize plants; Cadmium},
   language = {eng},
   issn = {0039-9140},
   journal = {Talanta},
   title = {Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection},
   volume = {79},
   year = {2009}
}

TY  - JOUR
ID  - 858780
AU  - Húska, Dalibor - Hubálek, Jaromír - Adam, Vojtěch - Vajtr, David - Horna, Aleš - Trnková, Libuše - Havel, Ladislav - Kizek, René
PY  - 2009
TI  - Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection
JF  - Talanta
VL  - 79
IS  - 2
PB  - Elsevier
SN  - 00399140
KW  - mRNA
KW  - Isolation
KW  - Magnetic particle and bead
KW  - Cyclic voltammetry
KW  - Square wave voltammetry
KW  - Brain tissue
KW  - Maize plants
KW  - Cadmium
N2  - 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.
ER  -

HÚSKA, Dalibor; Jaromír HUBÁLEK; Vojtěch ADAM; David VAJTR; Aleš HORNA; Libuše TRNKOVÁ; Ladislav HAVEL a René KIZEK. Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection. \textit{Talanta}. Elsevier, 2009, roč.~79, č.~2, 11 s. ISSN~0039-9140.

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