Evo m mlv rt mix kit with gdna clean for qpcr

Total RNA was isolated from the liquid-nitrogen-pulverized jejunal and ileal mucosa samples with the RNAiso Plus reagent (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s protocol. The concentration and quality of the RNA were determined using the NanoDrop One Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG11728, Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) following the manufacturer’s instructions. The RT-qPCR was then performed on a LightCycler R480II Real-Time PCR Instrument (Roche, Basel, Switzerland) using the SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) in accordance with manufacturer’s instructions. The fluorescence PCR program was set as follows: pre-denaturation, 95 °C for 30 s, 1 cycle; PCR amplification, 95 °C for 5 s, 60 °C for 30 s, 40 cycles; melting, 95 °C for 5 s, 60 °C for 1 min, 1 cycle; cooling, 50 °C for 30 s. Primers used in the PCR assay are listed in Table 1. The relative expression of selected genes normalized by β-actin was calculated using the 2^−ΔΔCt method [22 (link)]. The data were expressed as relative values to those for the CON-NBW group.

Twenty oocytes that were mixed into one sample were lysed in 100 μL RTL Lysis Buffer (containing 14.3 M β-mercaptoethanol and 1 μg/μL Carrier RNA). RNA was then extracted using the HiPure Total RNA Nano Kit (R4125-02, Magen, Guangzhou, China), and cDNA was synthesized using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG11728, Accurate Biology, Changsha, China). qPCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (AG11718, Accurate Biology, Changsha, China).
Based on the gene sequences of GPx3, CAT, Prdx3, Bcl2, Bcl-XL, and Caspase-3 from the National Center for Biotechnology Information, primers were designed using Primer 3 Plus software, and the primer information is shown in Table 1. The primers were all synthesized by the BGI-Huada Genomics company (Shenzhen, China).
The qPCR reaction system was 20 μL, comprised as follows: cDNA 1 μL, SYBR Mix 10 μL, each upstream and downstream primer 0.8 μL, ROX Low 0.4 μL, and ddH2O 7.8 μL. The qPCR reaction procedure was as follows: 95 °C for 3 s, 60 °C for 20 s, and 72 °C for 1 s. The relative expression of each gene was calculated using the 2^−ΔΔCt method [31 (link)].

Total RNA in liver samples was extracted with RNAiso Plus (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (Accurate Biotechnology (Hunan) Co., Ltd., Hunan, China) was used for reverse transcription. SYBR Green Premix Pro Taq HS qPCR Kit II (Accurate Biotechnology (Hunan) Co., Ltd., Hunan, China) and a quantitative thermal cycler (Roche LightCycler 96, Basel, Switzerland) were used for the RT-qPCR. The specific primers for lipid metabolism genes and reference genes are presented in Table 3. The amplification efficiency for all primers was 95~105%, and the coefficients of linear regression were >0.99. The PCR reaction system consists of 1 μL cDNA template, 0.4 μL forward primer (10 μM), 0.4 L reverse primer (10 μM), 5 μL SYBR Green Pro Taq HS Premix II, and 3.2 μL sterilized water. The program was as follows: 95°C for 30 s followed by 40 cycles of “95°C for 5 s, 57°C for 30 s, and 72°C for 30 s.” Melting curve analysis (1.85°C increment/min from 58°C to 95°C) was performed after the amplification phase for validation of a sole product. The mRNA expression was calculated with the 2^−ΔΔCt method [14 (link)].