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

Abstract

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 µL vs. 50 µL), 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 µL). 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.

MicroRNA-371a-3p—The Novel Serum Biomarker in Testicular Germ Cell Tumors

“…Whether serum or plasma should be used for measuring miR371 is an unresolved question. The present data indicate that both fluids can safely be employed [81]. However, the normalizer microRNA 30b-5p involves higher Ct values in plasma than in serum.…”

MicroRNAs for detecting occult genitourinary cancer

“…The blood processing method and type of sample can be vital when analyzing parameters such as miRNAs. For miR-371a-3p, there is a study showing little difference in miRNA levels between serum and plasma [63]. Despite this, to ensure accurate results, sample type should be universal and used only for miRNA extraction, if the test would enter the clinic in the near future [64].…”

MiRNA biomarkers in cancers of the male reproductive system: Are we approaching clinical application?

“…112 A recent analysis compared methods to isolate miR-371a-3p using 17 serum samples from TGCT patients and 10 serum and plasma control samples, showing that bead-based method was more stable and applicable on small volumes, but total RNA method exhibited higher sensitivity, due to a higher starting volume. 113 This is vital to choose the extraction method used in the future on clinical practice. Another important question relates to the choice of stable housekeeping miR-NAs (with levels of such miRNAs being found to vary between serum and plasma samples, with implications in sample type selection and laboratory processing 78 ) and also to whether the levels of the miRNAs can be evaluated only by measuring their cycle threshold values, or if this analysis needs an endogenous control in the reaction.…”

“…A recent analysis compared methods to isolate miR‐371a‐3p using 17 serum samples from TGCT patients and 10 serum and plasma control samples, showing that bead‐based method was more stable and applicable on small volumes, but total RNA method exhibited higher sensitivity, due to a higher starting volume 113 . This is vital to choose the extraction method used in the future on clinical practice.…”