Informace o publikaci
Utilization of paramagnetic microparticles for automatable serum RNA isolation
| Autoři | |
|---|---|
| Rok publikování | 2013 |
| Druh | Konferenční abstrakty |
| Citace | |
| Popis | 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. |