Real time pcr system c1000 thermal cycler

Total RNA was isolated from the mammary gland of dams by using the Total RNA Kit. The purity and concentration of RNA were measured by Nanodrop ultramicro spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 1 μg of RNA using the PrimeScriptTM RT reagent Kit. The mRNA expression was performed using a TB Green^® Premix Ex Taq™ II kit and Bio-Rad C1000 Thermal Cycler Real-Time PCR System (Bio-Rad, Hercules, CA, USA). Relative mRNA expression was measured by relative quantification with β-actin as the internal reference. The primer sequences of all the targets are shown in Table S2. Relative gene expression was calculated according to the 2^−ΔΔCt method.

The RNA samples for qPCR analysis were selected from the same colon tissues used for RNA sequencing. qPCR analysis was performed using a TB Green^® Premix Ex Taq™ II kit (Takara, Shanghai, China) and Bio-Rad C1000 Thermal Cycler Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The reverse transcription reaction system is a final volume of 10 μL, including 1 μg RNA, 1 μL PrimeScript RT Enzyme MixⅠ, 1 μL RT Primer Mix, 4 μL 5 × PrimeScript Buffer and RNase-free water (37 °C for 15 min and then 85 °C for 5 s). Amplification volume was 20 μL containing 2 μL cDNA, 0.8 μL forward primer (10 μM), 0.8 μL reverse primer (10 μM), 0.4 μL ROX Reference Dye (50×), 10 μL SYBR Premix Ex Taq Ⅱ and 6 μL RNase free water. The amplification conditions were a pre-denaturation program (95 °C for 30 s), and the amplification program (95 °C for 5 s, and 60 °C for 34 s) was for 40 cycles. The expression level of Gapdh was normalized [12 (link)]. Table S1 provides detailed information on the primers used. The relative expression levels of gene expression were calculated by the ΔΔCt method.