Preanalytic Methodological Considerations and Sample Quality Control of Circulating miRNAs

Abstract

As miRNAs emerge as potential circulating biomarkers for the diagnosis or prognosis of a wide variety of diseases, the quantification of miRNA necessitates careful preanalytic considerations and sample quality control becomes crucial. This study comprehensively analyzed the profiles of 356 miRNAs by quantitative RT-PCR in various blood sample types, with various processing protocols. The comprehensive analysis investigated the correlations of individual miRNAs with certain confounding factors. On the basis of these profiles, a panel of 7 miRNAs was established for the quality control of samples corresponding to hemolysis and platelet contamination. The panel was used to investigate the confounding impacts based on the size of the blood collection tube, the centrifugation protocol, post–freeze-thaw spinning, and whole blood storage. A standard dual-spin workflow for the processing of blood had been established for optimal sample quality. The real-time stability of 356 miRNAs was also investigated with demonstration of the temperature and time-induced miRNA degradation profile. Stability-related miRNAs were identified from real-time stability study and further incorporated into the quality control panel. This quality control panel enables the assessment of sample quality for more robust and reliable detection of circulating miRNAs. As miRNAs emerge as potential circulating biomarkers for the diagnosis or prognosis of a wide variety of diseases, the quantification of miRNA necessitates careful preanalytic considerations and sample quality control becomes crucial. This study comprehensively analyzed the profiles of 356 miRNAs by quantitative RT-PCR in various blood sample types, with various processing protocols. The comprehensive analysis investigated the correlations of individual miRNAs with certain confounding factors. On the basis of these profiles, a panel of 7 miRNAs was established for the quality control of samples corresponding to hemolysis and platelet contamination. The panel was used to investigate the confounding impacts based on the size of the blood collection tube, the centrifugation protocol, post–freeze-thaw spinning, and whole blood storage. A standard dual-spin workflow for the processing of blood had been established for optimal sample quality. The real-time stability of 356 miRNAs was also investigated with demonstration of the temperature and time-induced miRNA degradation profile. Stability-related miRNAs were identified from real-time stability study and further incorporated into the quality control panel. This quality control panel enables the assessment of sample quality for more robust and reliable detection of circulating miRNAs. miRNAs are 21- to 22-nucleotides long, evolutionarily conserved, noncoding RNAs that are found in eukaryotic cells, including in humans.1Lagos-Quintana M. Rauhut R. Lendeckel W. Tuschl T. Identification of novel genes coding for small expressed RNAs.Science. 2001; 294: 853-858Crossref PubMed Scopus (4023) Google Scholar Studies have subsequently found that miRNAs are involved in the posttranscriptional gene regulation of cellular processes, such as cell differentiation, proliferation, and apoptosis.2Hayes J. Peruzzi P.P. Lawler S. MicroRNAs in cancer: biomarkers, functions and therapy.Trends Mol Med. 2014; 20: 460-469Abstract Full Text Full Text PDF PubMed Scopus (1616) Google Scholar, 3Wahid F. Shehzad A. Khan T. Kim Y.Y. 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M. potential of and MicroRNAs in the of with high-risk Scopus Google Scholar Therefore, the confounding in miRNA M. V. S. M. J. E. G. G. of the the of on sample platelet and Scopus Google Scholar, B. Martin A. C. L. C. A. F. E. J. V. N. C. of collection and processing on plasma circulating from cancer Mol 20: Full Text Full Text PDF PubMed Scopus Google Scholar, K. of levels in plasma of and Scopus Google Scholar types of blood of blood processing may in cellular or hemolysis of plasma or in the miRNA that are to pathological J. Hu W. L. L. Y. Liu B. M. M. Zhu S. Liu S. of preanalytic on circulating microRNAs in whole 2014; PubMed Scopus Google Scholar platelet also a confounding of circulating Kim Y. Kroh E.M. Tewari M. processing circulating biomarker 2013; C. MicroRNAs in platelet and 2013; Full Text Full Text PDF PubMed Scopus Google Scholar The of miRNA detection and quantification in clinical reliable and Therefore, blood and processing A variety of studies have the of on the quantification of miRNAs in plasma and A. of circulating miRNAs in of and on their and Mol 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, The of on the of miRNA 7: Scopus Google Scholar, S.K. A. of circulating and 2011; PubMed Scopus Google Scholar studies the of and to stable miRNA Kim Y. Kroh E.M. Tewari M. processing circulating biomarker 2013; K. T. Y. S. C. S. M. K. Y. Y. T. The of on the analysis of circulating 7: PubMed Scopus Google Scholar In the was to workflow for blood processing to stable miRNA This study investigated the that to miRNA quantification in various A comprehensive analysis was by 356 miRNAs in various sample types established L. Zou R. 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