Reaction mixture

Total RNA was isolated using TRIZOL reagent (Invitrogen; Carlsbad, CA), and cDNA synthesis was carried out in a reaction mixture (Promega, Madison, WI) containing M-MLV Reverse Transcriptase, RNase inhibitor, dNTP, and random primers at 46 °C for 1 h. Quantitative real-time PCR on 96-well optical plates was performed in the qPCR Mastermix (Enzynomics, Daejeon, Korea), and fluorescence emitting from dye-DNA complex was monitored in CFX Connect Real-Time Cycler (BIO-RAD, Hercules, CA). The mRNA values of targeted genes were calculated relative to GAPDH expression. All reactions were performed in triplicate. The nucleotide sequences of PCR primers are summarized in Supplementary Table 3.

To characterize the SCCmec genes (I, II, III, IVa, V, and mecA), six primer pairs were used (Table 1). Briefly, 100 ng of pure DNA was added to a reaction mixture (Promega Corporation, Madison, WI, USA) containing 2.5 mM MgCl₂, 0.2 mM of each deoxynucleoside triphosphate (dATP, dGTP, dCTP, and dTTP), 1U of Taq DNA polymerase, varying concentrations of the different oligos and nuclease-free water. The genes were amplified under the following conditions: an initial denaturation step at 94°C for 5 min; 10 cycles of denaturation at 94°C for 45 sec, annealing at 65°C for 45 sec, and extension at 72°C for 1.5 min; 25 cycles of denaturation at 94°C for 45 sec, annealing at 65°C for 45 sec, and annealing at 55°C for 45 sec; and a final extension at 72°C for 10 min. The PCR products were separated by electrophoresis on 1.5% agarose gels and subsequently stained with 0.5 mg/mL ethidium bromide dissolved in 0.5X TBE buffer (Tris-borate-EDTA). The stained gels were visualized and analyzed under ultraviolet light. The SCCmec type I (S. aureus 1746), type II (S. aureus 1749), type III (S. aureus 1748), and type IVa (S. aureus USA 300 and MW2) strains were used as positive controls.

Total RNA was extracted from uterine tissues or stromal cells with the TRIzol solution (Invitrogen, CA) according to the manufacturer's protocol. RNA (2 µg) samples were reverse-transcribed into single-stranded cDNA in a 25 µl reaction mixture (Promega, China). Real-time PCR was then performed in a 20 µl reaction volume containing 10 µl of 2× Brilliant SYBR Green Mix (TaKaRa, China), 2 µl of template cDNA, 0.5 µM primers, and 300 nM reference dye using the ABI thermal cycler 7500. The thermal cycling conditions were 95°C for 30 sec, followed by 40 cycles at 94°C for 5 sec, 60°C for 34 sec. Melting curve analysis and agarose gel electrophoresis were conducted following the quantitative PCR assays to monitor PCR product purity. The results were analyzed using ABI Prism 7500 software (Applied Biosystems, USA). 18 S was employed for normalization. The following primers were used: Kiss1: sense, 5′-CGAAGGAGTTCCAGTTGTAGG-3′,antisense,5′-AAGGAATCGCGGTATGCA-3′;GPR54:sense,5′-CCGTCCAACGCTTCAGGAT-3′,antisense,5′-GTGTAGCGAAAAACAGGGGAA-3′; decidual prolactin-related protein (dPRP): sense, 5′-TTATGGGTGCATGGATCACTC C-3′, antisense, 5′-CCCACGTAAGGTCATCATGGA T-3;18S: sense, 5′-AATCAG GGTTCGATTCCG GA—3′, antisense, 5′-CCA AGA TCCAACTACGAGCT-3′.