Sds software 2

Total RNAs were extracted from 3 × 10⁶ tumor cells using RNeasy columns (Qiagen) and first strand cDNA was generated using a QuantiTect Reverse Transcription kit (Qiagen). PCR reactions were performed with gene-specific primers (Supplementary Table 1) using PCR Master Mix (Promega) in an Eppendorf Mastercycler PCR machine. For qRT-PCR, cDNA was amplified using RT² SYBR® Green ROX qPCR Mastermix (Qiagen) in a 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). Each amplification contained no-cDNA control wells and positive control wells using XpressRef™ Mouse Universal Total RNA (Qiagen). GAPDH was used as an internal normalization control. The data were analyzed using the SDS software 2.2.2 from Applied Biosystems.

RNA from semen cell sediments was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified with a NanoDrop device (NanoDrop Technologies, Wilmington, DE, USA). cDNA was synthesized from 500 ng of total RNA using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time quantitative PCR was performed on a 7900HT fast real-time PCR system using commercial Taqman assays (Applied Biosystems) for the following genes: Fn14, CD163, KLK2, KLK3, CXCR2 and CCR3. The cycle threshold (C_t) value for each sample was normalized to the expression of PPIA (cyclophilin A), which showed no significant gene expression differences between the compared groups of patients. SDS software 2.3 and RQ Manager 1.2 (Applied Biosystems) were used to analyze the results with the comparative C_t method (2^−∆∆Ct). All data were expressed as an n-fold difference relative to the calibrator (a mixture of the RNA from 4 different patients was used as a calibrator sample).

The expression by RT-qPCR of 6 miRNAs in the same 8 samples (n = 4 per group) (technical validation) or in 16 samples (the same 4 samples per group plus another 4 samples per group) (biological validation) were analyzed on the basis of their ratio ranking and their biological interest. It was using the TaqMan^® MicroRNA Reverse Transcription kit following the manufacturer's instructions (Thermo Fisher Scientific Inc., Waltham, MA). miRNA expression was assessed by real-time PCR using an Applied Biosystems 7500 Fast Real-Time polymerase chain reaction System (Applied Biosystems, Foster City, CA). Reactions were carried out in duplicate for all miRNAs using TaqMan™ MicroRNA Assay (Thermo Fisher Scientific Inc., Waltham, MA): hsa-miR-1-3p (477820_mir), hsa-miR-216a-5p (477976_mir), hsa-miR-31-3p (478012_mir), hsa-miR-20a-5p (478586_mir), hsa-miR-126-5p (477888_mir), and hsa-miR-204-5p (478491_mir). The threshold cycle value for each sample was normalized with the expression of hsa-miR-1-3p (Ref: 477820_miR). We chose this miRNA because it was the endogenous control tested with a more constant expression (with the lowest coefficient of variation) and with greater expression. SDS software 2.3 and RQ Manager 1.2 (Applied Biosystems, Foster City, CA) were used to analyse the results with the comparative Ct method (2^−ΔCt). The Log₂ FC of EVOO vs. SO were determined.

Mucor species quantification was performed on a 7900HT Fast Real-Time PCR System using the primers and probes described previously34 (link), with slight modifications. Mucor (F) 5′ GTC TTT GAA CGC AAC TTG CG 3′, Mucor (R) 5′ CCG CCT GAT TTC AGA TCA AAT T 3′ and Mucor probe: 5′ TTCCAATGAGCACGCCTGTT-MGBNFQ 3′. DNA from Mucor circinelloides (CBS277.49), belonging to the mold collection of the Fungal Biodiversity Center (CBS) was used as a positive control. For Mucor detection, PCR was performed in a final volume of 20 μl containing 0.5 μM of each primer of the Mucormycetes species, 0.25 μM of the internal control primers and 0.2 μM of Mucor spp. probe. For 18S (reference gene) amplification, we used primers, probes and conditions described previously35 (link). PCR conditions for Mucor spp., 18S and internal control were: 2 min at 50 °C for Uracil-DNA Glycosylase treatment, 10 min at 95 °C for Taq activation, 15 s at 95 °C for denaturation and 1 min at 61.2 °C for annealing and extension (50 cycles). SDS software 2.3 and RQ Manager 1.2 (Applied Biosystems) were used to analyze the results with the comparative threshold cycle (Ct) method (2^−∆∆Ct). C_t values for each sample were normalized with the 18S reference gene. All data were expressed as an n-fold difference relative to a calibrator (a mix of DNA from 4 human faecal samples).