title: Heparinase treatment of heparin-contaminated plasma from coronary artery bypass grafting patients enables reliable quantification of microRNAs.
abstract: BACKGROUND
  microRNAs have recently been identified as powerful biomarkers of human disease. Reliable polymerase chain reaction (PCR)-based quantification of nucleic acids in clinical samples contaminated with polymerase inhibitor heparin requires deheparinization. However, the effects of deheparinization procedure on quantification of nucleic acids remain largely unknown. The aim of this study was to determine whether the deheparinization procedure completely eliminates the inhibition of amplification, while maintaining RNA integrity and technical variability of the measured microRNA levels.
  
  
  METHODS
  Heparinized plasma from 9 patients undergoing coronary artery bypass grafting (CABG) and the heparin-free plasma from 58 rats were spiked with a synthetic RNA oligonucleotide and total RNA was extracted. The RNA solutions were then treated with heparinase I to remove contaminating heparin prior to reverse transcription. Levels of synthetic spike-in RNA oligonucleotide, as well as endogenous hsa-miR-1-3p and hsa-miR-208a-3p, were measured using quantitative reverse transcription PCR (RT-qPCR). The amplification efficiency and presence of inhibitors in individual samples were directly determined using calibration curves.
  
  
  RESULTS
  In contrast to RNA samples from rat plasma, RNA samples derived from the CABG patient plasma contained inhibitors, which were completely eliminated by treatment with heparinase. The procedure caused a decrease in the amount of detected RNA; however, the technical variability of the measured targets did not change, allowing for the quantification of circulating endogenous hsa-miR-1-3p and hsa-miR-208a-3p in the plasma of CABG patients.
  
  
  CONCLUSIONS
  The heparinase treatment procedure enables utilization of RT-qPCR for reliable microRNA quantification in heparinized plasma.
authors.affiliation: Institute of Molecular Biology and Genetics, Almazov Federal North-West Medical Research Centre, Saint-Petersburg, Russia.
authors.forename: Kirill
authors.initials: K
authors.lastname: Kondratov
authors.affiliation: Institute of Experimental Medicine, Almazov Federal North-West Medical Research Centre, Saint-Petersburg, Russia.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Kurapeev
authors.affiliation: Institute of Experimental Medicine, Almazov Federal North-West Medical Research Centre, Saint-Petersburg, Russia.
authors.forename: Maxim
authors.initials: M
authors.lastname: Popov
authors.affiliation: Department of Biotechnical Systems, Saint-Petersburg Electrotechnical University LETI, Saint-Petersburg, Russia; Saint-Petersburg State University, Saint-Petersburg, Russia.
authors.forename: Marina
authors.initials: M
authors.lastname: Sidorova
authors.affiliation: Institute of Experimental Medicine, Almazov Federal North-West Medical Research Centre, Saint-Petersburg, Russia.
authors.forename: Sarkis
authors.initials: S
authors.lastname: Minasian
authors.affiliation: Institute of Experimental Medicine, Almazov Federal North-West Medical Research Centre, Saint-Petersburg, Russia.
authors.forename: Michael
authors.initials: M
authors.lastname: Galagudza
authors.affiliation: Institute of Molecular Biology and Genetics, Almazov Federal North-West Medical Research Centre, Saint-Petersburg, Russia; Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden; Institute of Translational Medicine, ITMO University, Saint-Petersburg, Russia.
authors.forename: Anna
authors.initials: A
authors.lastname: Kostareva
authors.affiliation: Institute of Molecular Biology and Genetics, Almazov Federal North-West Medical Research Centre, Saint-Petersburg, Russia.
authors.forename: Anton
authors.initials: A
authors.lastname: Fedorov
keywords: Biomarkers; CABG, coronary artery bypass grafting; Cq, quantification cycle; EDTA, ethylenediaminetetraacetic acid; Heparinized plasma; PCR, polymerase chain reaction; RIN, RNA integrity number; RNase, ribonuclease; RT, reverse transcription; RT-qPCR efficiency; RT-qPCR, reverse transcription quantitative real-time PCR; TP, time point; cel-miR-39-3p, 3p strand of mature Caenorhabditis elegans microRNA-39; hsa-miR-1-3p, 3p strand of mature Homo sapiens microRNA-1; hsa-miR-208a-3p, 3p strand of mature Homo sapiens microRNA-208a; microRNA quantification; qPCR, quantitative real-time PCR; tRNA, transfer RNA