Abi 7300 fast real time pcr detection system

Total RNA was obtained from the mice liver using Trizol Reagent (TaKaRa, Dalian, China) and then reverse-transcribed using a commercial kit (Perfect Real Time, SYBR^® PrimeScript^™ TaKaRa, China) following the instructions of the manufacturer. The mRNA expression levels of specific genes were quantified via real-time PCR, using SYBR^®Premix Ex Taq^™ II (Tli RNaseH Plus) and an ABI 7300 Fast Real-Time PCR detection system (Applied Biosystems, USA). The SYBR Green PCR reaction mixture consisted of 10 μl SYBR^®Premix Ex Taq (2X), 0.4 μl of the forward and reverse primers, 0.4 μl of ROX reference dye (50X), 6.8 μl of ddH₂O and 2 μl of cDNA template. Each sample was amplified in triplicate. The fold-expression of each gene was calculated according to the 2^-ΔΔCt method [26 ], in which the β-Actin gene was used as an internal standard. The primer sequences used are given in Table 1.

Total RNA was obtained from meat tissue using Trizol Reagent (TaKaRa, Dalian, China), and then reverse-transcribed using a commercial kit (Perfect Real Time, SYBR^® PrimeScript™ TaKaRa, China) following the instructions of the manufacturer. The mRNA expression level of specific genes were quantified via real-time PCR, using SYBR^®Premix Ex Taq™ II (Tli RNaseH Plus), and an ABI 7300 Fast Real-Time PCR detection system (Applied Biosystems, USA). The SYBR Green PCR reaction mixture consisted of 10 μl SYBR^®Premix Ex Taq (2X), 0.4 μl of the forward and reverse primers, 0.4 μl of ROX reference dye (50X), 6.8 μl of ddH₂O, and 2 μl of cDNA template. Each meat tissue was amplified in triplicate. The fold-expression of each gene was calculated according to the 2^-ΔΔCt method [20 ], in which β-Actin gene was used as an internal standard. The primer sequences used are given in Table 1.

Total RNA was extracted from liver samples using the Trizol reagent (TaKaRa Biotechnology Co., Dalian, China), according to the manufacturer’s instructions. The concentration of RNA in the final preparations was measured with a Nanodrop ND-2000c spectrophotometer (Thermo Fisher Scientific, Camden, USA). Reverse transcription was immediately performed using the Primer-ScriptTM reagent Kit (Takara Biotechnology Co., Dalian, China).
The expression of genes in liver was measured using real-time quantitative PCR (RT-qPCR) with SYBR Premix Ex Taq II kit (Tli RNaseH Plus; Takara Biotechnology) and an ABI 7300 Fast Real-Time PCR detection system (Applied Biosystems). The 20 μL reaction system included 2 μL of cDNA template, 0.4 μL of ROX reference dye (50×), 10 μL of SYBR Premix Ex Taq (2×), 0.4 μL of each forward and reverse primer, and 6.8 μL of double-distilled H₂O. The RT-qPCR cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. All of the samples were run in triplicate, and the mRNA expression level of genes were calculated using the 2^−ΔΔCt method and normalized to the value of the reference gene β-actin. The final result of each target gene expression was expressed as the percentage of the CONT group. Primer sequences are shown in Table 2.