Izvorni znanstveni članak

https://doi.org/10.11613/BM.2020.010703

Study of the preanalytical variables affecting the measurement of clinically relevant free-circulating microRNAs: focus on sample matrix, platelet depletion, and storage conditions

Martina Faraldi ; Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto ortopedico Galeazzi, Milano, Italia
Veronica Sansoni ; Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto ortopedico Galeazzi, Milano, Italia
Silvia Perego ; Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto ortopedico Galeazzi, Milano, Italia
Marta Gomarasca ; Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto ortopedico Galeazzi, Milano, Italia
Jakub Kortas ; Department of Physical Culture, Gdańsk University of Physical Education & Sport, Gdańsk, Poland
Ewa Ziemann ; Department of Athletics, Strength and Conditioning, Poznan University of Physical Education, Poznan, Poland
Giuseppe Banfi ; Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto ortopedico Galeazzi, Milano, Italia; Università Vita-Salute San Raffaele, Milano, Italia
Giovanni Lombardi ; Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto ortopedico Galeazzi, Milano, Italia; Department of Physical Culture, Gdańsk University of Physical Education & Sport, Gdańsk, Poland

Sažetak

Introduction: Circulating microRNAs (miRNAs) are emerging as potential biomarkers. However, the lack of preanalytical and analytical standardization limits their use. The aim of this study was to determine the expression of different miRNAs in plasma according to different collection and storage conditions.
Materials and methods: Venous blood from 10 volunteers was collected in tubes spray-coated with dipotassium salt of ethylendiaminetetraacetic
acid, either with (plasma-preparation tube, PPT) or without (K2EDTA) gel separator. Platelet-poor plasma (PPP) was also obtained from K2EDTA plasma. After storage under different conditions, miRNA-enriched total RNA was isolated from plasma and reverse transcribed. A panel of 179 miRNAs was assayed by quantitative polymerase chain reaction and the results were analysed by GenEx software. Detectability and stability of miRNAs were determined.
Results: The number of undetected miRNAs was: 18, 24, and 22 in PPT; 83, 43, and 20 in K2EDTA; and 76, 106, and 104 in PPP samples, for plasma immediately frozen at - 80°C and plasma stored for 24h at room temperature or 4°C, respectively. Circulating miRNA expression in PPT samples was not affected by storage delay or temperature, while the percentage of up- and down-regulated miRNA in K2EDTA and PPP samples ranged from 2%, and 1% to 7%, and 5%, respectively.
Conclusions: Sample matrix, temperature and delay in storage strongly influence the expression level of plasma miRNAs. Our results indicate PPT tubes as the most suitable matrix to improve total miRNA detectability and stability, independently of temperature.

Ključne riječi

circulating microRNA; biomarkers; plasma; preanalytical phase

Hrčak ID:

234053

URI

https://hrcak.srce.hr/234053

Datum izdavanja:

15.2.2020.

Posjeta: 698 *