Staging of the cycle into estrus and diestrus was based on the cytology of the vaginal smear. All rats were killed between 10 00 a. m. and 12 00 noon and the uteri were collected and snap frozen in liquid nitrogen for storage at ?80 C until further analysis. Immature rats injected with gonadotropin selleck chemical were used for the contrac tion study. All procedures had been approved by the Committee on the Use of Live Animals for Teaching and Research, the University of Hong Kong. RT PCR of Imd, Crlr, and Ramps Total RNA of the uterus was obtained by homogenization in TRIZOL reagent using a polytron and subjected to RT PCR. RNA samples were reverse transcribed into complementary DNA with the SuperScript II reverse transcriptase. The real time RT PCR technique Inhibitors,Modulators,Libraries has been previously described.
Polymerase chain reactions were conducted Inhibitors,Modulators,Libraries by an iCycler iQ real time PCR de tection system using iQ SYBR Green Supermix. Three house keeping genes were tested and B actin was used as an internal standard based on its uniform expression across the groups. Standard curves for each primer pair were prepared by the serial dilution of cDNA to determine the PCR efficiency. The PCR efficiencies for Imd, Crlr, Ramp1, Ramp2, Ramp3 and Actb were all above 0. 95. The relative gene expression levels were then analyzed by The where Ct is the cycle threshold. The reaction mixtures contained 10 ul iQ SYBR Green Supermix, 2 ul template cDNA, 100 nM of each primer, and DNase free water to a final volume of 20 ul. Cycle conditions were 95 C for 5 min, followed by a maximum of 40 cycles of 95 C for 15 sec, 59 C for 15 sec, and 72 C for 15 sec, and extension at 72 C for 10 minutes.
The reaction was completed with a dissoci ation step for melting point analysis with 50 C to 95 C for 10 sec each. The design of the primers Inhibitors,Modulators,Libraries was based on the published sequences. Melt curve analysis for each primer showed only one peak for each product. The identities of all the PCR products Inhibitors,Modulators,Libraries were confirmed by gene sequencing. Measurement of IMD in the uterus Each tissue sample was homogenized in 3 ml 2N acetic acid and then boiled for 10 min. A 50 ul aliquot was taken for the protein assay and the remaining hom ogenate was centrifuged at 18600 X g for 20 min at 4 C. The supernatants were all lyophilized and stored at ?20 C until assay. The lyophilized tissue samples were reconstituted in 1X IMD assay buffer.
IMD level Inhibitors,Modulators,Libraries was measured with an IMD EIA kit. The minimum detectable concen tration was 0. 26 ngml and the range was 0 100 ngml. The intra Tofacitinib Citrate JAK assay and inter assay coefficients of variation were 10% and 15% respectively. The amount of protein in each sample was measured with a protein assay reagent spectrophotometrically at 595 nm. The immunoreactive IMD was expressed as pgmg protein. Gel filtration chromatography of the uterus The tissues were extracted with a polytron in 1N acetic acid on ice. A 50 ul aliquot of the homogenate was stored at ?20 C until protein assay.