title: Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material.
abstract: MicroRNAs (miRNAs) have emerged as promising cancer biomarkers. However, exploiting their informative potential requires careful optimization of their detection. Here, we compared the efficiency of commonly used RNA extraction kits in miRNA recovery from cells, plasma and urine/plasma-derived exosomes, using single-gene RT-qPCR and miRNA profiling. We used increasing amounts of starting material to investigate the impact of the input material size on miRNA extraction. We showed that miRNA recovery was largely influenced by the isolation method and by the amount of input material. In particular, the miRCURY™ kit provided highly pure RNA. However, its columns poorly recovered miRNAs from limiting amounts of cells and plasma, and rapidly saturated by large RNA species and plasma components, thus impeding miRNA recovery from high input amounts. Overall, the miRNeasy® kit permitted a better miRNA detection despite a less pure extracted RNA. Nevertheless, some miRNAs were preferentially or exclusively isolated by either of the methods. Trizol® LS resulted in very low purity RNA which affected RT-qPCR efficiency. In general, miRCURY™ biofluids kit efficiently extracted miRNAs from plasma. A careful selection of the RNA isolation method and the consideration of the type and size of input material are highly recommended to avoid biased results.
authors.affiliation: Department of Oncology, Luxembourg Institute of Health (LIH), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg.
authors.forename: Victoria
authors.initials: V
authors.lastname: El-Khoury
authors.affiliation: Department of Oncology, Luxembourg Institute of Health (LIH), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Pierson
authors.affiliation: Department of Oncology, Luxembourg Institute of Health (LIH), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg.
authors.forename: Tony
authors.initials: T
authors.lastname: Kaoma
authors.affiliation: Department of Oncology, Luxembourg Institute of Health (LIH), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg.
authors.forename: François
authors.initials: F
authors.lastname: Bernardin
authors.affiliation: Department of Oncology, Luxembourg Institute of Health (LIH), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg.|Centre Hospitalier de Luxembourg, 4 rue Barblé, L-1210 Luxembourg, Luxembourg.
authors.forename: Guy
authors.initials: G
authors.lastname: Berchem
mesh_terms: D015415:Biomarkers; D001826:Body Fluids / Q000737:chemistry; D045744:Cell Line, Tumor; D055354:Exosomes / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D035683:MicroRNAs / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D015415:Biomarkers; D035683:MicroRNAs