Abi prism viia7

Total RNA was isolated using the RNeasy Mini Kit. cDNA was synthesized using random hexamers (Invitrogen, Carlsbad, CA, USA) for subsequent real-time quantitative PCR analysis (ABI Prism VIIA7; Applied Biosystems Inc., Foster City, CA, USA). PCR products were detected using SYBR Green and normalized by cyclophilin expression. Primers were designed using Primer Quest (Integrated DNA Technologies, Inc., Coralville, IA, USA). Primer sequences were available upon request.

Total RNA was prepared using RNeasy Mini Kit (Qiagen), and first-strand cDNA was synthesized using the Retroscript Kit (Ambion) according to the manufacturer's instructions. Real time PCR was performed using the ABI Prism ViiA7 instrument (Applied Biosystems). NOS2 transcripts were quantified using TaqMan^® Pre-developed Assay Kits (Applied Biosystems) according to the manufacturer's instructions, and the results normalized to expression of GAPDH. The relative standard curve method was used to derive normalized expression for all samples, and “NOS2 fold induction” was calculated as the ratio of cytokine-treated samples to “LVS alone” or “Schu S4 alone” control samples.

Cells in 12-well plates were washed by phosphate buffered saline (PBS) three times, and then were given 0.5 mL TRIZOL reagent (Invitrogen, Carlsbad, USA). Tissues were pulverized and then given 1 mL TRIZOL reagent. Total RNA was extracted using the TRIZOL method. RNA (2 μg) was reverse transcribed to cDNA using a high-capacity cDNA reverse transcription kit (Promega Biotech, Madison, WI, USA). Gene expression was tested using real-time quantitative PCR analysis (ABI Prism VIIA7; Applied Biosystems) performed with a SYBR Green Master Mix (Promega Biotech, Madison, WI, USA) and normalized by cyclophilin expression. Primers were designed using Primer Quest (Integrated DNA Technologies, Coralville, IA, USA). Primer sets are described in Table 1.

Total RNA from whole ovary was extracted using a trizol reagent (15596018; Invitrogen). Reverse transcription of total RNA was performed with a high-capacity cDNA reverse transcription kit (R312-01/02, Vazyme). cDNA was diluted to 10 ng uL^-1. Real-time PCR analysis (ABI Prism VIIA7, Applied Biosystems) was performed with a SYBR Green Master Mix (Q511-AA, Vazyme) and normalized based on cyclophilin expression. The mRNA expression of related genes was normalized against that of Cyclophilina. The primers used for real-time PCR are shown in Table 1.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using random hexamers (Invitrogen, Carlsbad, CA,USA) for subsequent real-time quantitative PCR analysis (ABI Prism VIIA7; Applied Biosystems Inc, Foster City, CA,USA) according to the manual. PCR products were detected using Sybr Green and normalized by cyclophilin expression. Primers were designed using Primer Quest (Integrated DNA Technologies, Inc, Coralville, IA, USA). Primer sets for quantitative real-time PCR were summarized in Table 1.

For each sample DNA was extracted from whole blood using the AllPrep Isolation Kit (Qiagen, Hilden, Germany) or the Qiagen-mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. Blood was kept frozen before the extraction. Genotyping was performed using KASP (KBioscence, Hoddesdon, UK) and TaqMan (Thermo Fisher Scientific, Waltham, MA, USA) technologies. Genotyping was carried out using 384 well plates using 5ng of DNA for each sample. The order of DNA samples was randomized on plates in order to ensure that similar numbers of cases and controls were analyzed in each batch. Detection was performed using an ABI PRISM Viia7 sequence detection system with Viia7 software (Applied Biosystems, Foster City, CA, USA). The personnel performing the genotyping was blinded on the identity of the subject (i.e. whether the DNA belonged to a case or a control subject). For quality control, duplicates of 10% of the samples were interspersed throughout the plates. In addition, we discarded all the samples that had a call rate < 75%.