Enrichment of HLA-G-Specific miRNAs

To enrich HLA-G-specific miRs the recently published miTRAP method was employed [31 (link)]. Briefly, the complete 3′-UTR of HLA-G was cloned upstream of four MS2 loops, in vitro transcribed with Riboprobe (Promega, Mannheim, Germany) and used for the enrichment of HLA-G-specific miRs from cell lysates of the RCC cell line MZ2905RC (HLA-G mRNA⁺/ protein⁻). By application of 500 pmol of fusion protein consisting of the MS2 loop and maltose binding protein domains, in vitro-transcribed RNAs (HLA-G 3′-UTR and as a mock control a sequence encoding only the four MS2 loops) were loaded on amylose beads (NEB). After washing and blocking steps with yeast tRNA (Promega) and BSA (NEB), the beads were incubated with the cell lysate, then washed with wash buffer before the elution was carried out with maltose solution followed by RNA extraction with TRIzol Reagent (Invitrogen). A specific volume of the cell lysate was used for RNA extraction and applied as an input control. The miR enrichment in the eluates was validated by qPCR.