PMC:3475480 / 4464-9063 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3475480","sourcedb":"PMC","sourceid":"3475480","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3475480","text":"Methods\n\nAnimals\nBALB/c mice (Japan SLC, Inc., Shizuoka, Japan) were bred in a pathogen-free facility and used at 7 to 9 weeks of age for siRNA injection. The mice were maintained on a 12-h light/dark cycle at a constant temperature with free access to food and water. Every effort was made to minimize the number of animals that was used and their suffering per the Committee for the Care and Use of Laboratory Animals, College of Pharmacy, Kyung Hee University (KHP-2010-04-06).\n\nSynthesis of siRNA\nCasz1 siRNA oligonucleotides were synthesized by Genolution (Seoul, Korea). The target sequence for Casz1 was 5'-GATGTGATCCGACATTACA-3' (siRNA#1), 5'-CTAGAACTGCTCTGTATGT-3' (siRNA#2), and 5'-CAATTCGGCAAGTAATGGA-3' (siRNA #3), and nonspecific scrambled siRNA was used as a negative control.\nTo examine the efficacy of siRNAs, siRNAs were transfected into B16F10 cells using G-Fectin (Genolution); 1-2 µL of G-Fectin and 20 nM siRNA were added to 50 µL phosphate buffered saline, and the reaction was incubated for 10 min at room temperature (RT). B16F10 cells were used to seed 24-well plates at 5 × 104 cells/well and 30% confluence, and the transfection mixture was added to the plate. The cells were harvested for RNA extraction after 24 h of incubation. Total RNA was prepared using TRIzol (Invitrogen, Carlsbad, CA, USA) per the manufacturer's instructions.\n\nDelivery methods\nThe transfection agent was in vivo-jetPEI™ (Polyplus, Illkirch-Graffenstaden, France). Per the manufacturer, 25 µg siRNA and 6.5 µL vivo-jetPEI™ (N/P charge ratio of 6) were diluted with 50 µL 10% glucose solution and 50 µL sterile H2O. The solution was vortexed gently and left for 15 min at RT. The mixture was injected into the tail veins of 7- to 9-week-old mice, and the treated mice were measured for blood pressure or sacrificed for RNA extraction.\n\nRNA extraction and real-time PCR\nRNA was extracted from the tissues of siRNA-treated mice with TRIzol (Invitrogen). We synthesized cDNA from 500 ng of total RNA using the PrimeScript™ RT reagent kit (TaKaRa, Otsu, Japan) per the manufacturer's protocol. Casz1 mRNA was analyzed by real-time PCR. One-tenth of the cDNA reaction was added to a final volume of 20 µL for each reaction containing SYBR Green I (TaKaRa). The primers for Casz1 and glyceraldehye-3-phosphate dehydrogenase (GAPDH) were 5'-GTCTCTTCGGGAACTGCAAG-3'/5'-TGGGACACAGGCACTGTAGA-3' and 5'-GCTCTCTGCTCCTCCTGTTC-3'/5'-CAATACGACCAAATCCGTTG-3', respectively (forward/reverse sequence).\nQuantitative real-time PCR was performed on an ABI Step One Real-time PCR system (Applied Biosystems, Foster, CA, USA) using the following program: 45 cycles of 95℃ for 10 s, 60℃ for 15 s, and 72℃ for 20 s. To normalize the amount of sample cDNA in each reaction, the Ct value of each target gene was subtracted by that of GAPDH to obtain delta Ct [14]. To calculate the fold-change in expression, the delta Ct value of the case sample was subtracted by that of the control (delta delta Ct = delta Ct case - delta Ct control).\nReverse-transcription PCR was performed similarly to quantitative real-time PCR, except that it used 35 cycles for amplification. The PCR products were separated by electrophoresis in a 2.0% agarose gel and visualized under UV lamp after ethidium bromide staining.\n\nBlood pressure measurement\nBlood pressure was recorded intraarterially with a computerized data acquisition system (ADInstruments, Bella Vista, Australia). To place the intraarterial catheter, mice were anesthetized with avertin. The standard dose was 0.014 mL/g avertin, which was made by diluting stock solution (for stock solution, 10 g 2,2,2, tribromoethanol dissolved in 10 mL tertiary amyl alcohol) 40-fold in 0.9% NaCl. The intraarterial catheter, a polyethylene tube (0.2 mm I.D., 0.5 mm O.D.) (Natsume, Tokyo, Japan) that was filled with 0.9% NaCl containing 100 U/mL heparin, was inserted into the right carotid artery and tied in place.\nThe blood pressure in the vessel was transmitted along the catheter to the transducer's diaphragm (MLT0699 Disposable BP Transducer; ADInstruments). The diaphragm signal was amplified through a Bridge Amplifier and recorded on a PowerLab system (LabChart 7.2; ADInstruments). Blood pressure was monitored for 2 h after the injection of the anesthetic and calculated as the average blood pressure between 45 min and 75 min after the injection (1 point per min). Statistical analysis was performed using PASW Statistics version 18.0 (SPSS Inc., Chicago, IL, USA), and differences in blood pressure between case and control groups were analyzed by independent sample t-test.","divisions":[{"label":"Title","span":{"begin":0,"end":7}},{"label":"Section","span":{"begin":9,"end":480}},{"label":"Title","span":{"begin":9,"end":16}},{"label":"Section","span":{"begin":482,"end":1362}},{"label":"Title","span":{"begin":482,"end":500}},{"label":"Section","span":{"begin":1364,"end":1836}},{"label":"Title","span":{"begin":1364,"end":1380}},{"label":"Section","span":{"begin":1838,"end":3278}},{"label":"Title","span":{"begin":1838,"end":1870}},{"label":"Title","span":{"begin":3280,"end":3306}}],"tracks":[{"project":"2_test","denotations":[{"id":"23105927-11846609-44845687","span":{"begin":2836,"end":2838},"obj":"11846609"}],"attributes":[{"subj":"23105927-11846609-44845687","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#9593ec","default":true}]}]}}