Mastercycler gradient pcr apparatus

Hepatic total RNA was extracted from the liver tissue with TRIzol reagent in accordance with the manufacturer's instructions. First, RNA purity and concentration were tested using a nucleic acid/protein analyzer (Thermo, USA). Second, the extracted total RNA (1 μg) was reverse transcribed using the PrimeScript™RRT Reagent Kit with gDNA Eraser (TaKaRa Company, Dalian, China) on a Mastercycler gradient PCR apparatus (Eppendorf Company, Germany). Next, the complementary DNA was kept at −20°C before PCR amplification. Next, real-time polymerase chain reaction (PCR) analyses were performed in 48-well optical PCR plates using SYBR® Premix Ex Taq™ (TaKaRa Company, Dalian, China) on an Applied Biosystems StepOne Real-Time PCR System (Thermo, USA). Finally, 2^−ΔΔCT was used for analyzing the data. Primer sequences are listed in Table 1.

Total RNA was extracted from tissues using Trizol reagent (Servicebio, Wuhan, China). The purity and concentration of the total RNA were detected by the NanoDrop 2000 system (Thermo Scientific, Waltham, MA, United States). The cDNA was synthesized by reverse transcribing 2 μg of RNA using the Hifair^® Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen Biotech, Shanghai, China) in a Master-cycler gradient PCR apparatus (Eppendorf, Hamburg, Germany). Next, PCR was performed in a 20 μl volume containing 2 μl of cDNA, 8 μl of diluted and mixed primers, and 10 μl of SYBR Green Master Mix (Yeasen Biotech, Shanghai, China) using the LightCycler 96 System (Roche, Basel, Switzerland). The primers used are shown in Table 3 (Namvar et al., 2005 (link)), and the relative expression of HSV-2 gB mRNA was calculated using the Log10 (2^−△△Ct) method.

Pregnant mare serum gonadotropin (PMSG) was purchased from the Hangzhou Animal Medicine Factory, China. Human chorionic gonadotrophin (HCG) was provided by Livzon Pharmaceutical Factory, Zhuhai, China. Other materials included anti-vascular endothelial growth factor (VEGF) (Santa Cruz, SC-7269), anti-fibroblast growth factor 2 (FGF-2) (Immunoway, Cat NO.YT5549), anti-estrogen receptor α (Santa Cruz, SC-787); anti-progesterone receptor A (Proteintech, Cat NO.25871-1-AP); anti-LIF, (Gentex, Cat NO.GTX11940), ITG β-3(Abclonal, Cat NO.A2542) and Evans Blue (Cat. number E8010). Main reagents and devices included quantitative real-time PCR (qRT-PCR) equipment (Applied Biosystems, USA), SYBR Green qPCR Kit (Yesen, Cat NO.11201-11203), nucleic acid protein analyzer (DU730, Beckman Coulter, USA), Mastercycler gradient PCR apparatus (Eppendorf, Germany), Nikon microimaging system (TE2000-U, Tokyo, Japan), Step-One Real-Time PCR (Applied Biosystems, California, USA), scanning electron microscope (HITACHI, SU8100, Japan), estrogen ELISA kits (No.501890, Cayman, USA), progesterone ELISA kits (Cat: ELK7894, ELK Biotechnology, China), and Odyssey infrared imaging system (licor Biosciences, USA).