Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Inc. Valencia, CA, U.S.A.) at the 24 h ethanol treatment. Total RNA was reverse transcribed using the Reverse Transcription System (Promega, A3500, WI, USA) as per instructions. The primers used were as follows: Bax, (forward: 5'-AGGATGCGTCCACCAAGAAG-3') and (reverse: 5'-GCTCCCGGAGGAAGTCCAAT-3'), Bcl-2, (forward: 5'-GACTTCGCCGAGATGTCCAG-3') and (reverse: 5'-CATCCCAGCCTCCGTTATCC-3'), Fas, (forward: 5'-GGACCCTCCTACCTCTGGTTCTT-3') and (reverse: 5'-CAGTCCCTAGCTTTCCTTTCACC-3'), and GAPDH, (forward: 5'-CGTGGAAGGACTCATGAC-3') and (reverse: 5'-CAAATTCGTTGTCATACCAG-3'). 30 cycles polymerase chain reaction (PCR) was performed for amplification of these products with each cycle, which consists of denaturation at 94℃ for 30 s, annealing at Tm (melting temperature) for 30 s and primer extension at 72℃ for 1 min. PCR products were separated on 1.5% agarose gel (Bioline, BIO-41025, London, U.K.) and were visualized by RAS-3000 (FUJI, RAS-3000, Japan).
Ras 3000
Manufactured by Fujifilm
Sourced in Japan
The RAS-3000 is a film processor from Fujifilm. It is designed for automatic development of various types of photographic film.
Automatically generated - may contain errors
Lab products found in correlation
Proteome profiler array, by R&D Systems (1 mentions)
Triton x 100, by Biosesang (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Reverse transcription system, by Promega (1 mentions)
3 protocols using ras 3000
1
Quantitative Gene Expression Analysis
2
Decellularized Tissue Cytokine Profiling
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Cytokines for adipogenesis and angiogenesis in the adipose and heart dECMs were investigated using a microarray kit (Proteome Profiler Array, R&D Systems, Minneapolis, MN, USA). Native and decellularized tissue samples were treated with 1% (v/v) Triton X-100 (Biosesang, Seongnam-si, Korea) in PBS with protease inhibitors and chopped for digestion. After homogenization, freezing, thawing, and centrifugation for 5 min to remove cell debris, supernatants containing cytokines were added to each array membrane on a shaker overnight at 4 °C. After treatment with the detection antibody, a Chemi Reagent Mix was added to the membranes. Finally, the membranes were exposed using an X-ray imaging machine (RAS-3000, Fuji, Aichi, Japan).
3
Western Blot Analysis of RGMa Protein
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Rat spinal cords were lysed using 150 mM NaCl, 1% Triton X-100, 20 mM HEPES (pH 7.4), 10% glycerol, 5 mM EDTA, and complete protease inhibitor cocktail (Roche Applied Science). The lysates were clarified by centrifugation at 15,000 g at 4 °C for 30 min, and the supernatants were collected and subjected to SDS-PAGE. They were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% bovine serum albumin in PBS containing 0.05% Tween-20 for 1 h and incubated with anti-RGMa antibody in blocking solution. After washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1: 3,000, Cell Signaling Technology) for 1 h. Detection was performed using Pierce Western Blotting Substrate Plus (Pierce) and RAS-3000 (Fuji Film).
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