Individual taqman mirna assays

Individual TaqMan miRNA assays (Applied Biosystems) were performed according to the manufacturer’s instructions. Total RNA (10 ng in 5 µl per sample) isolated from EV was converted to cDNA using the microRNA reverse transcriptase Kit (Applied Biosystems) with 3 µl of specific miRNA assay RT primer in a reaction volume of 15 µl. The cDNA was setup in triplicate qRT-PCRs containing 1 µl of specific TaqMan miRNA assay and run on Viia7 Real Time PCR System (Applied Biosystems). Data were analysed using the ΔΔCt method with dnIKK2-Treg-EV as the reference and snRNA U6 as the endogenous control. Specific TaqMan miRNA assays used in this study were: U6 snRNA ID 001973, mmu-miR-293 ID 001794, hsa-miR-330-5p Assay ID 002230, mmu-miR-503 Assay ID 002456, hsa-miR-9 Assay ID 00058, hsa-miR-126-5p Assay ID 000451, rno-miR-207 Assay ID 001315, mmu-miR-297c Assay ID 002480, hsa-miR-484Assay ID 001821.

The expression levels of representative miRNAs, differentially expressed in the first trimester maternal plasma of women who later experienced sPTD as compared to the control group were verified in an independent cohort consisting of 58 plasma samples (n = 29 sPTL and n = 29 controls) using qRT-PCR. cDNA synthesis and qRT-PCR were performed using a TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Inc., Foster City, CA 94404, USA) and individual TaqMan MiRNA assays (Applied Biosystems, Inc., USA), following the manufacturer’s recommendations. All reactions were run in triplicate in a LC480 LightCycler system (Roche GmbH, Rotkreuz, Switzerland). The miRNA gene expression was determined using the 2^−ΔΔCt method [42 (link)]. RNU44 (Applied Biosystems, Inc., Foster City, CA 94404, USA) was used for normalization purposes.

Expression measurements of selected targets (mature miR-34a and miR-601) were studied in all tumor (n = 19) and control samples used in the initial miRNA microarray analysis and in an additional blinded independent set of 30 snap-frozen embryonal tumor samples (n = 21 MBs and n = 9 AT/RTs) using qRT-PCR. In brief, cDNA synthesis and subsequent RT-PCR were performed using a TaqMan MiRNA Reverse Transcription kit (Applied Biosystems, Inc., USA), and individual TaqMan MiRNA assays (Applied Biosystems, Inc., USA), according to the manufacturer’s recommendations. All samples were tested in triplicate in a LC480 LightCycler system (Roche GmbH, Switzerland). The RNU44 was used as endogenous control. Wells lacking template were used as negative controls. An additional online available miRNA microarray MB dataset from Feretti et al. was used, with GEO accession number GSE12303³ [23 (link)]. Expression changes were compared by relative quantification in the form of fold changes obtained with the ΔΔCt method.