Cfx384 real time pcr

Total RNA extracted from fresh brain tissues was reverse transcribed using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) with 2 µg of input RNA and random primers (Thermo Fisher Scientific, USA). qRT-PCR reactions were performed in 384-well plates using specific primers (Tm = 60 °C) (TIB MOLBIOL, Berlin, Germany) (Table 1) and iTaq SYBR Green Supermix (BioRad, Hercules, CA, USA) as a fluorescent detection dye, in CFX384TM Real-Time PCR (BioRad, USA), in a final volume of 10 µL. To characterize generated amplicons and to control contamination by unspecific by-products, melt curve analysis was applied. Results were normalized to β-actin or Gapdh mRNA level. All reactions were performed in duplicate, and results were calculated using the ∆∆Ct method.

Total RNA of liver, abdominal fat, and hepatocyte samples was isolated with RNAiso Plus (TaKaRa, Code no. 9109, Dalian, China), following manufacturer’s instructions. The total RNA concentration of each sample was measured by determining absorbance at 260 nm (Thermo Fisher Scientific, NanoDrop 2000, Waltham, USA) and RNA integrity was evaluated by agarose gel electrophoresis. Then, RNA samples were reverse-transcribed to complementary DNA (cDNA) according to the manufacturer’s instructions (PrimerScriptTM RT Master Mix, TaKaRa, Code no. RR036A, Dalian, China), as follows: incubation at 37 °C for 15 min, followed by 85 °C for 5 s. Quantitative Reverse Transcription (qRT) PCR analysis was performed in a fluorescence detection system (Bio-Rad CFX384TMReal-Time PCR, USA), with these cycling conditions: 30 s at 95 °C for denaturation, 5 s at 95 °C, 34 s at 60 °C (40 cycles), 15 s at 95 °C, and a final step for 1 min at 60 °C. Primers were designed using Primer Premier 6 software (Table S1), and synthesized commercially (Tsingke Biotech, Beijing, China). Beta-actin was the internal reference gene, due to its stable expression in Pekin ducks (Anas platyrhnchos). The PCR amplification efficiency for each primer ranged from 90% to 110%, with each sample analyzed in triplicate. Relative mRNA expression of target genes was determined using the 2^−ΔΔCt method [30 (link)].

Microarray results were confirmed by qRT-PCR. Total RNA extracted from fresh brain tissue was reverse transcribed using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, MA, USA) with 2 μg of input RNA and random primers (Thermo Fisher Scientific, MA, USA). qRT-PCR reactions were performed in 384-well plates using specific primers (Tm = 60°C) (TIB MOLBIOL, Germany) (see S1 Table) and the iTaq SYBR Green Supermix (BioRad, CA, USA) as a fluorescent detection dye, in CFX384TM Real-Time PCR (BioRad, CA, USA), in a final volume of 10 μl. To characterize generated amplicons and to control contamination by unspecific by-products, melt curve analysis was applied. Each reaction was performed in triplicate. All results were normalized to Gapdh mRNA level and calculated using the ΔΔCt method.