Sybr premix ex taq tli rnase h plus qpcr kit

Based on the manufacturer's instructions, Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from tissue samples. The ratio of OD260: OD280 was 1.8-2.0 in all samples. The integrity of RNA was detected by agarose gel electrophoresis. Based on the manufacturer's guidelines, Moloney murine leukemia virus (Promega, Madison, WI, USA) was used to reverse transcribe RNA samples into complementary DNA.
The SYBR® Premix Ex Taq (Tli RNaseH plus) qPCR kit (TaKaRa Biotechnology, Inc., Shiga, Japan) and ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) were used for qPCR detection. The primers are listed in Table 2. As mentioned earlier, the amplification efficiency of each primer was determined by using the double continuous dilution of cDNA (32 (link)). The amplification efficiency of GAPDH was nearly 100%. The magnification outcomes were confirmed by two methods: agarose gel electrophoresis and sequencing. As mentioned earlier, the relative expression of target gene mRNA was analyzed by the 2^−ΔΔCt method (33 (link)). Gene expression was normalized to GAPDH and presented as relative fold change compared to the NC group. All samples were analyzed in triplicate.

Total RNA was extracted from ovaries using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The complementary DNA was synthesized using the PrimeScript RT reagent kit with gDNA eraser (TaKaRa, #RR047A). The quantitative real-time PCR was performed using the SYBR Premix Ex Taq (Tli RNase H Plus) qPCR kit (TaKaRa, #RR420A) and 7500 Real-Time PCR system (Applied Biosystems). The expression of the target genes relative to the housekeeping gene (β-actin) was analyzed by the 2^−ΔΔCT method of Livak and Schmittgen [19 (link)]. Relative mRNA abundance of each target gene was normalized to the control group. Primer sequences are given in Table 2.

Total RNA was extracted from jejunal mucosa using TRIzol reagent (Invitrogen), according to the manufacturer's instructions. Total RNA (1 μg) was reverse transcribed with PrimeScript RT reagent kit with gDNA eraser (TaKaRa, #RR047A) for quantitative RT-PCR with SYBR Premix Ex Taq (Tli RNase H Plus) qPCR kit (TaKaRa, #RR420A) and 7500 Real-Time PCR system (Applied Biosystems). The relative mRNA abundance of the target genes was standardized with β-actin as the invariant control and was analyzed by the 2^−ΔΔCT method of Livak and Schmittgen. Primer sequences are given in Supplementary Table 2.