75 ml of Isogen (Nippon Gene Co Ltd , Tokyo, Japan) and then mix

75 ml of Isogen (Nippon Gene Co. Ltd., Tokyo, Japan) and then mixed Combretastatin A4 in vivo thoroughly with 0.15 ml of chloroform. The mixture was centrifuged (20,000 × g for 5 min), and then the aqueous phases were collected,

and 0.4 ml of isopropanol was added. The precipitated total RNA was recovered and washed with 70% (v/v) ethanol. The purity and concentration of the total RNA thus obtained were confirmed using an Experion electrophoresis system (Bio-Rad Laboratories, Inc., California, USA) and a NanoDrop 1000 click here spectrophotometer (Thermo Fisher Scientific K. K., Massachusetts, USA). Construction of gene specific primers Gene specific primers were designed by using Primer-BLAST (http://​www.​ncbi.​nlm.​nih.​gov/​tools/​primer-blast/​). The primers used were as follows: for ATPGD1 (NM_134148), forward primer, 5′-CCCTGGCCTTCGACCTCTCTCCAT-3′ and reverse primer, 5′-CGGCACTGGGGCCCATCCTTC-3′ to yield a 164-bp product; for CN1 (NM_177450), forward primer, 5′-TGGTGGCATCCTCAACGAACCA-3′

and reverse primer, 5′-TCCAGGAATTAGGATGTGGCCTGA-3′ to yield an 88-bp product; for ß-actin (NM_007393), forward primer, 5′-ATGAGCTGCCTGACGGCCAGGTCATC-3′ and reverse primer, 5′-TGGTACCACCAGACAGCACTGTGTTG-3′ to yield a 192-bp product. Quantification of mRNA levels cDNA was synthesized by using a PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, Inc., Shiga, Japan). The genomic DNA in the RNAs extracted from tissues was eliminated with gDNA Eraser, which were then reverse-transcribed selleck chemicals by PrimeScript RT. Each 25 μl of the PCR reaction mix contained a 2 μl template, 0.2 μM of each primer, and 1× ROX Reference Dye II in 1× SYBR Premix Ex Taq

II (Takara Bio, Inc.). The reaction was performed at 95°C for 30 s; this was followed by 40 cycles at 95°C for 5 s and at Amobarbital 60°C for 20 s. The fluorescence was measured at the end of the extension step in each cycle. Following cycling, a melt curve analysis was performed after each quantitative PCR to ensure that a single product had been amplified per primer set. The fold-change of the gene expression was calculated using the 2-∆∆Ct method with ß-actin as an internal control. Student’s t-test was used (P < 0.05 or P < 0.01) to test statistical significance. Detection of carnosine in muscle and blood Vastus lateralis muscle samples were deproteinized with 1 ml of 5% (w/v) sulfosalicylic acid. The samples were centrifuged at 20,000 × g for 5 min, and then the supernatants were filtered with a 0.45-μm filter. Blood samples were dissolved in 1 M perchloric acid (final concentration, 0.3 M) and centrifuged at 20,000 × g for 5 min. KOH (3 M) was added to the supernatants to realize a final concentration of 4.25% v/v. After centrifugation (20,000 × g for 5 min), the obtained supernatants were filtered and applied to a TSKgel ODS-80Ts column (Tosoh Co., Tokyo, Japan) equilibrated with 4% (v/v) acetonitrile, 100 mM sodium 1-pentanesulfonate, and 200 mM ammonium dihydrogen phosphate (pH 2.0).

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