Get access

Utilization of paramagnetic microparticles for automated isolation of free circulating mRNA as a new tool in prostate cancer diagnostics

Authors

  • Michaela Fojtu,

    1. Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Jaromir Gumulec,

    1. Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
    2. Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Jan Balvan,

    1. Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
    Search for more papers by this author
  • Martina Raudenska,

    1. Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
    Search for more papers by this author
  • Marketa Sztalmachova,

    1. Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
    2. Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic
    Search for more papers by this author
  • Hana Polanska,

    1. Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
    Search for more papers by this author
  • Kristyna Smerkova,

    1. Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic
    Search for more papers by this author
  • Vojtech Adam,

    1. Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
    2. Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic
    Search for more papers by this author
  • Rene Kizek,

    1. Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
    2. Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic
    Search for more papers by this author
  • Michal Masarik

    Corresponding author
    1. Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
    2. Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
    • Correspondence: Dr. Michal Masarik, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech Republic

      E-mail: masarik@med.muni.cz

      Fax: +420-5-4949-4340

    Search for more papers by this author

  • Colour Online: See the article online to view Fig. 4 in colour.

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.

Get access to the full text of this article

Ancillary