Total RNAs from rat hepatocytes, HepG2 cells and mouse liver were prepared by using a STRAIGHTFORWARD BLUE whole RNA extraction kit. Simple strand cDNA synthesis was done using 5 mg of oligo dT primers, RNA and reverse transcriptase in a volume of 50 ml. PCR reactions were conducted in 20 ml consisting of 2 ml of the cDNA ATP-competitive Chk inhibitor product, 0. 2 mM of each dNTP, 20 pmol of each primer and 0. 8 units of Taq polymerase. PCR was performed at 95 8C for 30 s, followed by annealing for 30 s, and 72 8C for 1 min. The past cycle was accompanied by a extension phase at 72 8C for 10 min. The RT PCR products and services were electophoresed in 0. 8% agarose ties in under 100 V and were stained with 0. 5 mg/ml ethidium bromide. Checking densi tometry was performed with i MAXTM Gel Image Analysis System. Levels of the house keeping genes were used to correct for variations in RNA degradation, RNA isolation and the efficiency of the reverse transcription. Real time PCR was performed using 1 ml of cDNA in a ml reaction volume with all the LightCycler real time PCR System. The double stranded DNA certain dye SYBR Green I was integrated into the PCR buffer provided in the SYBR Premix Ex Taq reagent. The temperature profile of the reaction was 95 8C for 15 min, followed by 30 cycles of denaturation at 95 8C for 30 s, and extension at 72 8C for 1 Organism min. A family member gene expression quantification method was used to assess the change of mRNA expression according to the comparative threshold cycle method using house being an endogenous control keeping genes. The primers and annealing conditions for both methods are shown in Dining table The animal research project was reviewed and approved by the Institutional Animal Ethics Committee of Kyung Hee Universi ty. Five week previous ICR mice were housed in a temperature and humidity controlled room using a period of 12 h light/12 h darkness and free access to water and food. Mice were randomly divided into the following four groups : a regular diet fed group, a higher fat diet fed group and two therapy groups fed a plus oral administration of BA at 5 mg/kg bodyweight or 10 mg/kg. The body fat was measured twice weekly. After 3 weeks of therapy with BA, livers were removed, weighed and frozen straight away in liquid nitrogen. Liver cells Decitabine ic50 were homogenized in a answer of chloroform and methanol and incubated at 4 8C overnight following the addition of 50 mM sodium chloride. After centrifugation, the lipid fractions were dried with nitrogen and the total lipid content was measured. Next, the dried lipids were dissolved in 10 % Triton X 100 in PBS, and the triglyceride levels were measured according to the manufacturers directions for Triglyceride Reagents.