Malignant germ cell tumors, also known as germ cell cancers (GCC), are the most frequent cancer in young Caucasian males, with an increasing incidence during the last decades. They are clinically and histologically subdivided into seminomas and nonseminomas. The last can be composed of different elements, including embryonal carcinoma (the stem cell component), teratoma (TE) (somatic lineage) as well as yolk sac tumor (YST) and choriocarcinoma (CHC) (extra-embryonic lineages). The currently used serum markers, informative for both diagnosis as well as follow-up are AFP and hCG, predominantly indicative for the presence of the YST and CHC components. Recently, we reported that a selected set of miRNAs are highly expressed in all histological elements of GCC, with the exception of TE, including the miR 371-3 cluster. In addition to the presence in the primary cancers, they can also be identified in the serum of patients with such a malignancy, outperforming the classical markers AFP and hCG (Gillis and Rijlaarsdam et al. Molec. Oncol. 2013; Rijlaarsdam et al. Andrology, 2015). Here we demonstrate the power of the TaqMan Advanced miRNA Assays (PN 478007, Applied Biosystems) in combination with the use of the TaqMan miRNA ABC Purification Kit (PN 4473087, Applied Biosystems), starting with 50 μL human serum. The protocol includes polyadenylation of the miRNA targets at the 3’ end, followed by an adapter ligation at the 5’ end. Universal forward and reverse primers are used for amplification, followed by detection using the Applied Biosystems 7500 RealTime PCR System. Quality controls were added using dilution experiments of the cell lines, also included as inter-plate control, as well as for cDNA synthesis and RT-PCR efficiency. The non-human spike-ins ath-miR159a and cel-miR-39-3p were added as exogenous controls, while hsa-miR-30b-5p was used as endogenous control for normalization (reference). In total 30 GCC-related sera as well as 10 normal controls proved the informativity as well as efficiency of the developed pipeline in a standardized laboratory setting.

Citation Format: Leendert HJ Looijenga, Ton Van Agthoven, Ad Gillis. Efficient detection of germ cell cancer related miRNAs in serum using the TaqMan miRNA ABC purification bead kit in combination with the Taqman advanced miRNA assays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1088.