Sybr green mix

The expression pattern of miR-130b was analyzed in cDNA samples obtained from granulosa cells, immature and matures oocytes and their corresponding cumulus cells, and preimplantation embryos using quantitative real time PCR (qPCR). For this, mature miRNA-130b specific primers were purchased from Qiagen (Hilden, Germany). The qPCR was performed by mixing 2.5 μl template cDNA with 12.5 μl of SYBRGreen mix (Qiagen, Valencia, CA), 10× miScript Universal Primer and 10× miScript Primer assay in 25 μl of final volume. The qPCR was performed for 45 cycles of 95 °C for 15 s and 60 °C for 1 min in 7000 Real-Time PCR system (Applied Biosystems, USA). The threshold cycle (C_t) values of miR-130b and the endogenous controls were recorded using Sequence Detection Software (SDS v1.2.1, Applied Biosystems, USA). The qPCR data were normalized using the geometric mean of U6 and small nuclear RNAs (Snord48). The miRNA expression levels were then determined from the triplicate runs using the 2^-ΔΔCt method. All experiments were performed at least in three biological triplicates.

The expression levels of three tomato pathogenesis-related genes (PR-1, PR-2, and PR-3) and one polyphenolic gene (CHS) were analyzed using the qPCR and normalized to the reference β-actin gene. Primer nucleotide sequences used in this investigation are listed in Table 2. For each biological sample, the experiments were repeated three times. As previously described (Abdelkhalek et al., 2018 ), qRT-PCR was carried out using a QIAGEN Rotor-Gene 6,000 (ABI System, United States) with Thermo SYBR Green Mix (Foster, CA, USA). The relative expression level of the tested gene was calculated according to the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)).

Media was removed from plates and constructs were washed twice with PBS before the addition of 500 μL of TRIzol^® (Life Technologies, Paisley, United Kingdom). Each construct was then homogenized and the RNA extracted according to the manufacturer's instructions. Reactions were prepared in triplicate in 96-well plates and consisted of 70 ng of RNA diluted in 9.5 μL of nuclease-free water, 0.15 μL of both forward and reverse primers at a final concentration of 750 nM (see Table 1 for primer sequences), 0.2 μL of the QuantiFast Reverse Transcriptase Kit (Qiagen, Crawley, United Kingdom), and 10 μL of SYBR Green Mix (Qiagen). Plates were transferred to an Mx3005P Thermal Cycler for one-step RT-PCR, which was programmed to perform the following: 10 min at 50°C (reverse transcription), 5 min at 95°C (Hot Start Taq polymerase), followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Fluorescence was detected at the end of each cycle and data were analyzed using the 2^(−ΔΔCT) method using POLR2β as a reference gene and a single aneurally cultured construct from each experiment as a calibrator.

After obtaining the hub gene through the co-expression network, we verified it by RT-PCR. We collected 10 peripheral blood sample sure, including 5 normal controls and 5 pre-eclampsia blood samples. Follow the manufacturer’s recommendations to synthesize cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Trans, Beijing). SYBR Green Mix (QIAGEN, Germany) and specific primer sets were used to perform RT-PCR on the obtained cDNA. The PCR product was verified by melting curve analysis. The delta-delta Ct method was used to calculate the relative quantitative expression, and each gene was normalized to GAPDH. Three biological replicates and three technical replicates were used for analysis. The primers and primer sequences of each gene are shown in supplementary information 1.

The hepatic total RNAs were isolated with TRIzol reagent and reversely transcribed to the cDNAs with commercial cDNA synthesis kit. The mRNA levels of inflammatory cytokines were determined by qRT-PCR with the specific primers and SYBR Green mix from QIAGEN on the LightCycler^® 480 System from Roche Diagnostics International AG (Rotkreuz, Switzerland). The mRNA levels of different biomarkers were normalized against the mRNA levels of β-actin.