Bullseye evagreen qpcr 2x master mix

Samples with RNA integrity values >6.0 and absorbance 260/280 between 1.8 and 2.4 were used in this study. 0.4 microgram of RNA was reverse transcribed into cDNA using a mix of random hexamer primers (High Capacity cDNA Synthesis Kit, Life Technologies, Carlsbad, CA, USA). Quantitative polymerase chain reaction assays (qRT-PCR) were performed using ViiA7 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Each 25 microliters reaction contained Bullseye EvaGreen qPCR 2X Mastermix (MIDSCI, St. Louis, USA) and primers at a concentration of 0.05 mM. Primer sequences are indicated in Table 2. Amplification steps used were as follows: denature at 95°C for 10 min, annealing at 95°C for 15 s extension at 60°C for 45 cycles of amplification and 95°C extension for 15 s Melting curve was performed using the following conditions: 60°C for 1 min following by 5°C/s and 95°C for 15 s qRT-PCR assays were performed in triplicates. The geometric mean of the two reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin beta (ACTB), were used to normalize for input RNA. Expression data was analyzed using the comparative ΔΔCt method. In this method, the amount of target is normalized to an endogenous reference and relative to a calibrator.