Comparative Analyses of Liquid-Biopsy MicroRNA371a-3p Isolation Protocols for Serum and Plasma

Simple Summary The active disease status of patients with a malignant germ cell tumor can be evaluated using detection of specific body-circulating microRNAs. However, various methods are reported to isolate and detect microRNAs from blood, possibly influencing the score as positive or negative. Here, we investigated two frequently used techniques for microRNA isolation from blood, either serum or plasma, to evaluate possible differences. These data are required to compare published studies and to select the best methods in the future. No effect of either starting with plasma or serum was found, indicating that both blood products can be used. The bead-based method was more stable and applicable on small blood volumes, whereas the total RNA method exhibited a higher sensitivity due to a larger starting volume. These results are important to develop the optimal method for the detection of microRNAs in blood to monitor malignant germ cell tumor patients in clinic practice. MicroRNAs (miRNAs) are short, non-coding RNAs involved in translation regulation. Dysregulation has been identified in cancer cells. miRNAs can be secreted and detectable in body fluids; therefore, they are potential liquid-biopsy biomarkers. The miR-371a-3 cluster members are an example, monitoring the presence of malignant germ cell tumors based on patient serum/plasma analyses. However, a large variety of isolation techniques on sample types (serum vs. plasma) are reported, hampering interstudy comparisons. Therefore, we analyzed the impact of using the miRNeasy Serum/Plasma Kit (cell-free total RNA purification) Qiagen extraction kit and the TaqMan anti-miRNA bead-capture procedure of ThermoFisher for miRNA isolation. Ten normal male matched serum and plasma samples and seventeen testicular germ cell tumor patient serum samples were investigated. The Qiagen kit requires a higher input volume (200 mu L vs. 50 mu L), resulting in higher sensitivity. Serum and plasma comparison demonstrated high similarity in miRNA levels. Titration experiments showed that the bead-capture procedure is superior in cases of lower starting volumes (<100 mu L). This study highlights the strengths and limitations of two different isolation protocols, relevant for in vivo analysis with small starting volumes. In summary, miRNA detection levels results varied little between plasma and serum, whereas for low volumes the bead capture isolation method is preferable.

Automated summary

The study found that isolating microRNAs from plasma or serum had little impact on the results, and both blood products can be used. The bead-based method is more stable and suitable for small samples, while the total RNA method is more sensitive due to larger starting volume.