|Public on May 05, 2012
|Moderate Restricted Carbohydrate Diet Supplemented Mouse 2
|Moderate Restricted Carbohydrate Diet Supplemented Mouse
tissue: liver tissue (non-tumor region)
disease model: human -HrasG12V / shp53/ GFP4 transposon vector induced transgenic mouse with liver cancer
|Following a preliminary feeding of each diet formula for one week, HrasG12V / shp53 / GFP4 gene containing transposon vector were injected into mouse tail vein by hydrodynamic injection method. After 5 weeks of diet supplementation, all mice were sacrificed. Mouse liver tissue was excised for microarray analysis.
|Five- week- old male C57BL6 mice were randomly divided into two groups (n=3/group) and fed control diet (control group, LabDiet, Brentwood, MO, USA) or moderate restriced carbohydrate diet formula (MCD, Treat group) in a specific pathogen free zone. All procedures were approved by the institutional animal use and care committee.
|Non-tumor liver region was isolated from each mouse liver tissue. Total RNA extracted using Trizol following manufacturer's instructions.
|Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|1.65 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565AA) using one color scan setting.
|Agilent Feature Extraction Software (v 126.96.36.199) was used for background subtraction and LOWESS normalization.
|May 04, 2012
|Last update date
|May 05, 2012
|250 seonsan-ro seodaemum-gu
|Analysis of differential genetic expression by moderate-carbohydrate restriction diet with calorie restriction mimetic multiple phytochemicals (MCDmp) using a transgenic liver cancer model developed by a simple hydrodynamic transfection method.