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Supplemental Data (Wang et al.)

Supplemental Data Methods

RNA isolation of seminal plasma and serum

For the Solexa sequencing assay of the seminal plasma, an equal volume of seminal plasma from each participant was pooled separately to form cases and control sample pools.TRIzol reagent (Invitrogen) was used to extract total RNA from each pool of seminal plasma samples (120 mL each). The aqueous phase was subjected to 3 steps of acid phenol/chloroform purification to eliminate protein residues before isopropyl alcohol precipitation. The resulting RNA pellet was dissolved in 30 L RNase-free water and stored at 80°C until further analysis.

For the RT-qPCR assay,an amount of 100 L of seminal plasma or serum was mixed with 200 L acid phenol, 200 L chloroform, and 300 L RNase-free water. The mixture was vortex-mixed vigorously and incubated at room temperature for 15 min. After phase separation, the aqueous layer was mixed with 1.5 volumes of isopropyl alcohol and 0.1 volumes of 3 mol/L sodium acetate (pH 5.3). This solution was stored at -20°C for 1 h. The RNA pellet was collected by centrifugation at 16,000g for 20 min at 4 °C. The resulting RNA pellet was washed once with 750 ml/L ethanol and dried for 10 min at room temperature. Finally, the pellet was dissolved in 20 L of RNase-free water and stored at -80°C until further analysis.

Solexa sequencing of seminal plasma miRNAs

Solexa sequencing was performed asfollows. Briefly, after PAGE purification of small RNA molecules (<30 nucleotides) and ligation of a pair of Solexa adaptors to the RNA 5′ and 3′ ends, RNA molecules were amplified using primers to the adaptor regions for 17 cycles. Fragments that were approximately 90 bp (small RNA+adaptors) were isolated from an agarose gel. Purified DNA was used for cluster generation and sequencing analysis using the Illumina’s Solexa Sequencer according to the manufacturer’s instructions. Image files were generated by the sequencer and were processed to produce digital-quality data. After masking the adaptor sequences and the removal of contaminated reads, clean reads were processed for in silico analysis as previously described(12).

Quantification of miRNAs by hydrolysis-based RT-qPCR

Hydrolysisprobe-based RT-qPCR assayof serum or seminal plasma was performed according to the manufacturer’s instructions (Applied Biosystems) with a minor modification. Briefly, the reverse transcription reaction was carried out in 10 μL containing 2 μL of extract RNA, 1 μL of 10 mmol/L dNTPs, 0.5 μL of AMV reverse transcriptase (TaKaRa) , 1 μL of a stem-loop RT primer (Applied Biosystems), 2 μL of 5×reverse transcription buffer and 3.5 μL of diethylpyrocarbonate (DEPC) water. For synthesis of cDNA, the reaction mixtures were incubated at 16 oC for 30 min, at 42 oC for 30min, at 85 oCfor 5 min, and then held at 4oC. Real-time PCR was performed (1 cycle of 95 oC for 5 min, and 40 cycles of 95 oC for 15 sec and 60 oC for 1 min) with an Applied Biosystems 7300 Sequence Detection System. The reaction was performed with a final volume of 20 μL containing 1 μL of cDNA, 0.3 μL of Taq, 0.33 μL of hydrolysis probe (Applied Biosystems), 1.2 μL of 25 mmol/L MgCl2, 0.4 μL of 10 mmol/L dNTPs, 2 μL of 10×PCR buffer, and 14.77 μL of DEPC water. All reactions, including no-template controls, were performed in triplicate. The resulting Cq values were determined using fixed threshold settings and the absolute concentration of each target wascalculated by the calibration curve.

Theconcentration of sperm miRNAwas also determinedby a hydrolysisprobe-based RT-qPCR assayafterreverse transcriptase using specific RT primers (TaqmanMicroRNA Assay, Applied Biosystem). For reverse transcription, 80 ng of total RNA was used per cDNA preparation in a 10 μL RT reaction system including 1 μL of 10 mmol/L dNTPs, 0.5 μL of AMV reverse transcriptase (TaKaRa) , 1 μL of a stem-loop RT primer (Applied Biosystems), 2 μL of 5×reverse transcription buffer and 3.5 μL of diethylpyrocarbonate (DEPC) water.The procedure and RT-qPCR parametersfor the synthesis of cDNA of sperm were the same as the parameters forserum or seminal plasma (described above).All reactions were performed in triplicateand the absolute concentration of each target wascalculated by the calibration curve.

For each assay, calibration curves were prepared byten-fold serial dilution of synthetic single-strand miRNAsfor the seven target miRNAs (including miR-34c-5p, miR-122, miR-146b-5p, miR-181a,miR-374b, miR-509-5p, and miR-513a-5p, synthesized by TaKaRa, Dalian, China) from 10 fM/L to 105 pM/L, and the levels of the synthetic miRNAs were assessed by RT-qPCR assay. The resulting Cq values were plotted versus the log10 of the amount of the synthetic miRNAs. Each sample and each dilution of the calibrators were run in triplicate for analysis. All data were collected and analyzed with an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA).Every batch of amplifications included three water blanks as negative controls for each of the reverse transcription and the PCR steps.Employing the standard curves to calculate theabsolute concentrations of seven serum miRNAs. The limit of detection and dynamic range for each miRNA was was 102 fM/L and 102 fM/L～1×104 pM/L (hsa-miR-34c-5p), 10 fM/L and 10 fM/L～1×104pM/L (hsa-miR-122), 10 fM/L and 10 fM/L～1×105 pM/L (hsa-miR-146b-5p), 102 fM/L and 102 fM/L～1×104 pM/L (hsa-miR-181a), 10 fM/L and 10 fM/L～1×104 pM/L(hsa-miR-374b), 10fM/L and 10 fM/L～1×104 pM/L (hsa-miR-509-5p), and 10 fM/L and 10 fM/L～1×104 pM/L (miR-513a-5p).Semilogarithmic plots of the calibration curves for various concentrations of the synthetic single-strand miRNA calibrators were linear from 10 fmol/L to 105 pmol/L.

Assay precision of hydrolysis probe-based RT-qPCR assay

FortherepeatabilityofRNA extraction, pooled seminal plasma from 20 healthy persons was divided into two identical portions (each 500L),and RNA was extracted from the two pooled seminal plasma samples, respectively. Then the amounts of sixteen miRNAs (including miR-1, miR-106b, miR-122, miR-128, miR-146b-5p, miR-181a, miR-193a-3p, miR-19b, miR-34c-5p, miR-374b, miR-495, miR-509-5p, miR-513a-5p, miR-532-5p, miR-890, miR-9) ranging from low to high levels were measured with hydrolysis probe-based qRT- PCR assay. Each sample was run in triplicate for analysis. Threshold cycle (Cq) values of replicate assays were similar (r2=0.969), indicating that the RNA extraction method was reproducible.

The analytical repeatability of the hydrolysis probe-based RT-qPCR assay，exclusive of RNA extraction,was evaluated by detecting the same twenty miRNAs as above in the two same RNA preparations extracted from pooled serum collected from twenty healthy individuals.Each sample was run in triplicate for analysis.As a result, the analytical reproducibility of the RT-qPCR assay was good (r2=0.978).

The assay precision of the miRNA quantification, inclusive of RNA extraction, was evaluated by respectively performing 20 replicates of miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-374b, miR-509-5pand miR-513a-5pquantification.The mean CVs for the RT-qPCR assays (including the RNA extraction step) for these miRNAs were 6.0%,12.2%, 6.8%, 11.2%, 8.9%, 8.2%, and 10.1%, respectively.