Abi quantstudio 7 flex real time detection system

Total RNA of the same sources and concentrations with library preparation was reverse transcribed into cDNA using PrimeScript RT Reagent Kit (TaKaRa, Japan) following the manufacturer's instruction. Quantitative RT-PCR was performed on ABI QuantStudio 7 Flex Real-time Detection System (Life Technologies Holdings Pte Ltd, USA). Each 10.0 μL PCR mixture contained 5 μL of SYBR Premix Ex Taq™ II, 0.5 μL (10 pM) of each primer, 0.2 μL of ROX Reference Dye II (50 × ), 1.5 μL of cDNA (100 ng), and 2.3 μL of ddH₂O. Thermo cycling conditions consisted of an initial denaturing at 95°C for 3 min, for 40 cycles of amplification (95°C for 30 s and 60°C for 34 s), followed by thermal denaturing (95°C for 15 s, 60°C for 60 s, and 95°C for 15 s) to generate melting curves to verify amplification specificity. Pigeon β-Actin was used as an endogenous control. Relative quantifications of genes were calculated by 2^−ΔΔCT method. The primers of randomly selected genes and lncRNAs were designed using Prime3 and NCBI Primer-Blast.