Rnaprep pure micro kit

Porcine granulosa cells were harvested and extracted for total RNA using an RNAprep pure MicroKit (Aidlab, RN07, Beijing, China) according to the manufacturer's protocols. And the cDNAs were synthesized using a TURE script first strand cDNA Synthesis Kit (Aidlab, PC1802, Beijing, China) as described [34 (link)]. The reaction program was: 40 min at 42°C, 65°C for 15 min, and finally a cooling step at 4°C. For RNA-Seq, control and treatment granulosa cells were collected after in vitro ZEA exposure for 72 h. In each group, we applied 3 independent samples to extract total RNA using TRIZOL and sequenced at Novogene (Beijing, China).

The total RNA was extracted from MEFs and MCF7 with the RNA prep pure Micro Kit (Aidlab, RN07, Beijing, China). TransScript One‐Step gDNA Removal and cDNA Synthesis SuperMix were used to make the cDNA (TransGen, AT311‐03, Beijing, China). QuantStudio 5 was used to perform RT‐qPCR amplification using specified primers (Table S2) (Applied Biosystems, CA, USA). The relative transcript abundance was determined using the 2^−ΔΔCt method and normalized using the housekeeping gene actin as a reference.

As previously described (Wang Y. F. et al., 2018 (link)), the total RNA was isolated from six ovary tissues with the RNAprep Pure Micro Kit (Aidlab, RN07, Beijing, China). The cDNA was synthesized by TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, AT311-03, Beijing, China). Quantitative real-time PCR (RT-qPCR) amplification with specific primers (Supplementary Table 2) was performed using LightCycler 480 (Roche, Germany). The relative transcript abundance was calculated by the 2^–ΔΔCt method and normalized according to the housekeeping gene Gapdh.

The extraction of total RNA from tissues was performed with RNAprep pure Micro Kit (Aidlab, RN07, Beijing, China) according to the manufacturer’s instructions and then reverse-transcribed into cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, AT311-03, Beijing, China). Thermal cycler program was set as 50 min at 42 °C, 65 °C for 15 min, and finally a cooling step at 4 °C. Real time quantitative PCR (RT-qPCR) was performed using a Light Cycler real-time PCR instrument (LC480; Roche, Basel, Switzerland) using Light Cycler SYBR Green I Master Mix (Roche, 04887352001) according to the manufacturer’s instructions. The primers (Table S2) used were designed with Primer Express software (Applied Biosystem) with β-actin used as housekeeping positive control for amplification, the reactions of which were performed in 20 μl reaction volume containing 2 μl cDNA, 10 μl of SYBR green master mix, 0.4 μl of each primer forward and reverse gene (20 μM), and 7.2 μl of nuclease-free water per sample. The PCR conditions were as follows: 10 min at 95 °C, followed by 35 cycles at 95 °C for 10 s, 60 °C for 30 s and finally a cooling step at 4 °C. Each sample had 3 technical replicates, and the reactions were performed in triplicate for each gene. Gene expression changes were analyzed by the 2^−△△Ct method and normalized to β-actin.

Total RNA was extracted using the RNAprep pure Micro Kit (Aidlab, RN28, Beijing, China) according to the manufacturer’s instructions. Reverse transcription was performed using the TransScript One-Step gDNA Removal Kit and cDNA Synthesis Kit (TransGen Biotech, AT311, Beijing, China). All primers used in this research are listed in Supplementary Table S1. Relative quantification analysis was carried out with the LightCycler 480 II (Roche) using the LightCycler 480 SYBR Green I Master Kit (Roche, 04887352001) according to the manufacturer’s instructions. Each sample contained three technical replicates and reactions were performed with three biological replicates. The PCR conditions were as follows: 10 min at 95 °C, followed by 45 cycles of 95 °C for 10 s, 60 °C for 30 s and 72 °C for 20 s. Gene expression levels were calculated using β-actin for normalization. Relative transcript abundance was calculated using the 2^-ΔΔCT method.^{41 (link)} Data were expressed as mean±SD and calculated from at least three times independent replicates.

Ovaries of mice under different light-dark cycles (exposed for 4, 8 and 12 days) were collected. Total RNA was extracted using RNAprep pure MicroKit (Aidlab, RN07, China). Reverse transcription was performed using TURE script first strand cDNA Synthesis Kit (Aidlab, PC1802). Quantitative real-time PCR (qRT-PCR) was carried out using Light-Cycler^® SYBR Green I Master Kit (Roche, 04887352001, Switzerland) with a Roche real time PCR instrument (Roche LC480) according to the manufacturer’s instructions. qRT-PCR primers were listed in Table 1. The PCR reaction programs were set as follows: 10 min at 95°C, followed by 45 cycles of 95°C for 10 s, 60°C for 30 s and 72°C for 20 s. The relative mRNA abundance was normalized to the expression of β-actin according to following formula: 2^{−(target gene C}_T ^{value − reference gene C}_T ^value). Data represented were calculated by mean ± standard deviation (S.D.) of at least three independent experiments.

Total RNA was extracted using RNAprep pure MicroKit (Aidlab, Beijing, China) following the manufacturer’s instructions. cDNA was synthesized by using cDNA Synthesis Kit (TaKaRa, Dalian, China). Reverse transcription parameters were set as follows: 42 °C for 50 min and 65 °C for 15 min. For PCR, the primers were listed in table 1. PCR amplification was carried out on a Light Cycler real-time PCR instrument (Roche, Mannheim, Germany). The amplification procedure was conducted according to manufacturer’s instructions using a Light Cycler SYBR Green I Master (Roche). The PCR parameter were set at 95 °C for 10 min, followed by 55 cycles at 95 °C for 10 s, 60 °C for 30 s, and finally cooling at 4 °C.