title: Utilization of paramagnetic microparticles for automated isolation of free circulating mRNA as a new tool in prostate cancer diagnostics.
abstract: Determination of serum mRNA gained a lot of attention in recent years, particularly from the perspective of disease markers. Streptavidin-modified paramagnetic particles (SMPs) seem an interesting technique, mainly due to possible automated isolation and high efficiency. The aim of this study was to optimize serum isolation protocol to reduce the consumption of chemicals and sample volume. The following factors were optimized: amounts of (i) paramagnetic particles, (ii) oligo(dT)20 probe, (iii) serum, and (iv) the binding sequence (SMPs, oligo(dT)20 , serum vs. oligo(dT)20 , serum and SMPs). RNA content was measured, and the expression of metallothionein-2A as possible prostate cancer marker was analyzed to demonstrate measurable RNA content with ability for RT-PCR detection. Isolation is possible on serum volume range (10-200 μL) without altering of efficiency or purity. Amount of SMPs can be reduced up to 5 μL, with optimal results within 10-30 μL SMPs. Volume of oligo(dT)20 does not affect efficiency, when used within 0.1-0.4 μL. This optimized protocol was also modified to fit needs of automated one-step single-tube analysis with identical efficiency compared to conventional setup. One-step analysis protocol is considered a promising simplification, making RNA isolation suitable for automatable process.
authors.affiliation: Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
authors.forename: Michaela
authors.initials: M
authors.lastname: Fojtu
authors.forename: Jaromir
authors.initials: J
authors.lastname: Gumulec
authors.forename: Jan
authors.initials: J
authors.lastname: Balvan
authors.forename: Martina
authors.initials: M
authors.lastname: Raudenska
authors.forename: Marketa
authors.initials: M
authors.lastname: Sztalmachova
authors.forename: Hana
authors.initials: H
authors.lastname: Polanska
authors.forename: Kristyna
authors.initials: K
authors.lastname: Smerkova
authors.forename: Vojtech
authors.initials: V
authors.lastname: Adam
authors.forename: Rene
authors.initials: R
authors.lastname: Kizek
authors.forename: Michal
authors.initials: M
authors.lastname: Masarik
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood* / Q000302:isolation & purification; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D006801:Humans; D059346:Magnets; D008297:Male; D008863:Microspheres; D008875:Middle Aged; D015336:Molecular Probe Techniques; D011471:Prostatic Neoplasms / Q000175:diagnosis*; D012333:RNA, Messenger / Q000097:blood* / Q000302:isolation & purification; D055815:Young Adult
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger
keywords: Metallothionein; Microseparation; Paramagnetic microparticles; Prostate cancer; Serum mRNA