a 2013

Utilization of paramagnetic microparticles for automatable serum RNA isolation

GUMULEC, Jaromír a Michal MASAŘÍK

Základní údaje

Originální název

Utilization of paramagnetic microparticles for automatable serum RNA isolation

Autoři

Vydání

XIII. Pracovní setkání fyzikálních chemiků a elektrochemiků, 2013

Další údaje

Typ výsledku

Konferenční abstrakt

Utajení

není předmětem státního či obchodního tajemství
Změněno: 13. 12. 2013 11:40, Mgr. Veronika Dvořáková, Ph.D.

Anotace

V originále

Fast, easily automatable and simple procedure for isolating circulating mRNA from limited amounts of plasma/serum is highly required. Promising in this regard could be use of paramagnetic nanoparticles. Therefore, we compared isolation made by paramagnetic microparticles with methods commonly used for RNA isolation (silica columns and Trizol RNA Isolation). Another aim was to optimize serum isolation protocol using SMPs and to reduce the need of chemicals and sample volume. The amount of (a) magnetic particles, (b) oligo (dT)20 probe, and (c) serum volume were optimized. Consequently, RNA content was measured, expression of metallothionein was analyzed to demonstrate measurable RNA content and ability for real-time PCR detection. Isolation is possible on broad range of serum volumes (10–200 microL) without distinct efficiency, or purity decrease. For effective RNA isolation, the amount of SMPs can be reduced up to 5 microL, however, the best results are achieved using 10–30 microL SMPs. The volume of oligo (dT)20 does not affect isolation efficiency, when used in range 0.1–0.4 microL. In addition, this optimized protocol was modified to fit needs of automated one-step single-tube analysis. No significant differences in MT Ct values were observed between conventional and modified one-step analysis. Thus, one-step analysis protocol is considered a promising simplification making RNA isolation suitable for automatable process.