All subjects gave written informed consent before participation in the study. Genomic DNA was extracted from circulating leucocytes. The 4b4a genotype was identified directly after a polymerase chain reaction (PCR) amplification protocol,10 and the −786T>C and 894G>T genotypes were identified by PCR, followed by restriction fragment length polymorphism. Quality control for these assays was assessed by randomly selecting samples to be re-genotyped by 2 independent researchers. No misgenotyping was observed. Haplotypes for each subject were inferred using the software PHASE version 2.1 (University of Washington, Seatle,
Wash).21 The volunteers and researchers were blinded to the genotypes and haplotypes during the study. The PCR was performed using the following
Selleck Cabozantinib primers: sense 5’-AGG CCC TAT GGT AGT GCC TTT-3’ and antisense 5’-TCT CTT AGT GCT GTG GTC AC-3’. DNA was amplified for 35 cycles consisted of denaturing at 94°C GSK-3 beta phosphorylation for 30 seconds, annealing at 51°C for 40 seconds, and extension at 72°C for 40 seconds. The PCR products were digested by incubation with a restriction endonuclease, NgoMIV (New England Biolabs, Ipswich, MA), at 37°C for 16 hours and visualized by gel electrophoresis. The PCR was performed using the following primers: sense 5’-AGG CCC TAT GGTAGT GCC TTT-3′ and antisense 5′-TCT CTT TAG TGC TGT GGT CAC-3′. DNA was amplified for 35 cycles, and each cycle was composed of denaturing at 94°C for 1 minute, annealing at 49°C for 40 seconds, and
extension at 72°C for 40 seconds. The PCR products ID-8 were visualized by gel electrophoresis. The PCR was performed using the following primers: sense 5’ AAG GCA GGA GAC AGT GGA TGG A-3’ and antisense 5’ CCC AGT CAA TCC CTT TGG TGC TCA-3’. DNA was amplified for 35 cycles consisted of denaturing at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 1 minute. The PCR products were digested by incubation with Ban II (New England Biolabs, Ipswich, Mass) at 37°C for 16 hours and visualized by gel electrophoresis. Blood was drawn in the morning after 12 hours of fasting. Cholesterol and its subfractions (high-density lipoprotein [HDL] and LDL], as well as triglycerides, were determined by the dry chemistry method. Plasma glucose was measured by enzymatic in vitro test. The experimental protocol was conducted in the morning, 1 hour after a standardized light breakfast. The evaluation was performed from the first to the 12th day after the onset of menstruation. Subjects did not drink alcohol or caffeinated beverages and did not perform intense physical activities for at least 24 hours before the experimental visit. During the study, subjects were placed in a supine position in a quiet air-conditioned room (≈24°C), and they rested quietly for 10 minutes before any measurement.