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Lab Report Essay

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MATERIAL AND METHODS Drugs and reagents Minoxidil was purchased from Sigma (St Louis, MO, U.S.A) and dissolved in 0.12 mM HCl at concentration of 1.0 mg, then stored at -20°C. Human platelet lysates (HPL) preparation HPLs generated from platelet units were obtained from Iranian Blood Transfusion Organization. Thrombocyte concentrates had a platelet count of 3×1011 in 200 mL plasma collected with apheresis. Platelet units were frozen twice at −80°C and rethawed at 37°C to obtain platelet-released growth factors. The lysates were centrifuged at 2600 ×g at 4 ºC for 30 min to remove membrane fragments and then supernatant was filtered through 0.2 µm PVDF syringe filters (Jet – Biofil, Canada). 2 U/mL heparin was added to HPLs and stored at −80°C …show more content…

The mRNA concentration was quantified using a NanoDrop Spectrophotometer (Thermo Scientific, USA). The cDNA was synthesized with 1 µg of total RNA using the Revert Aid H Minus First Strand cDNA Synthesis Kit (Fermentas) and subjected to PCR amplification in a final volume of 25 µl. Primer set, annealing temperature, number of cycles, and PCR product length are shown in Table 1. The PCR products were electrophoresed on 1.2% agarose gel, and the bands were visualized on a UV transilluminator (Uvidoc, UK). GAPDH was used as an internal normalization …show more content…

Primers were blasted in NCBI website: http://www.ncbi.nlm.nih.gov/tools/primer-blast. Quantitative Real-time PCR was performed using Power SYBR Green PCR Master Mix (EURx, Ltd, Gdan˜sk, Poland) and Real-Time PCR System (Roche, Applied Science, Mannheim, Germany). Each reaction mixture contains a total volume of 20μl (master mix 10μl, cDNA 4μl, primer 2μl, and H2O 4μl). Cycling started with denaturation at 95°C for 10 min, followed by 50 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s and extension at 72°C for 10 s. The GAPDH mRNA level was used as an internal control to normalize the RNA levels from different samples. The Ct data was determined using default threshold settings. The fold changes normalized to GAPDH, were determined using the calculation of the relative quantitation (2−ΔΔCt). The equation used to calculate fold changes was as follow: ΔCt = Ct(sample) – Ct(GAPDH) and ΔΔCt = (ΔCt(sample)

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