Abi prism 7900 fast sequence detection system

RT−PCR was used to validate the potential therapeutic targets which predicted by network pharmacology. EESCs were seeded in 6-well plates and divided into control group and SZC group, The SZC group was treated with 200 µg/ml SZC and incubated at 37°C for 24 h. Total RNA was extracted form the EESCs using the TRIzol^® reagent (Invitrogen, 15596018). The RNA was reverse-transcribed into cDNA utilizing a Prime Script RT Reagent Kit (Takara, RR036A). RT−PCR was performed using SYBR Premix Ex Taq (Takara, RR820A), and the results were analyzed using a ABI Prism 7900 Fast Sequence Detection System (Thermo Fisher Scientific). Each sample was analyzed in three replicate wells, and the fold change in the transcriptional expression of the above genes was calculated using the 2−ΔΔCt method. The relative mRNA expression levels were normalized to those of ACTB. The primer sequences of these genes are provided in Supplementary Table S1.

Total RNA was extracted from human or murine placental tissues or cells by using Fast Tissue RNA Purification Kit (EZBioscience, EZB-RN5), which was then reverse-transcribed into cDNA with 4×Reverse Transcription Master Mix (EZBioscience, A0010GQ). Quantitative reverse transcription-PCR (RT-qPCR) was performed using 2× Color SYBR Green qPCR Master Mix (ROX2 plus) (EZBioscience, A0012-R2) and the analysis was based on ABI Prism 7900 Fast Sequence Detection system (Thermo Fisher Scientific). The fold change at the transcriptional level of the target genes was displayed by calculating the 2^-ΔΔCt method and each sample was run in triplicates. Relative mRNA expression levels were normalized to β-actin (ACTB). Sequences of each pair of primers was displayed in Table 1.
Moreover, EZ-press Tissue Direct PCR Kit (EZBioscience, EZB-TDP1) was used to genotype Il-27ra allele of dissected murine embryos as manufacturer's instructions. PCR products were then amplified with the following primers: Wild-type Forward: 5′-CGCAGAAAGTTCTCATCT-3′; Wild-type Reverse: 5′- TCATACAGTACCCATCCC-3′; Knockout Forward: 5′- ACCGTAAAGCACGAGGAA-3′. Knockout Reverse: 5′- GACCACCAAGCGAAACAT -3′. Next, 2% agarose gel in TBE buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA) was prepared for electrophoresis at 90 V for 60 min.

Total RNA was isolated with Qiagen RNA extraction kit according to the manufacturer’s protocol. One microgram of total RNA was used for reverse transcription reaction using iScript Reverse Transcription kit (Bio-rad Hercules, CA) according to manufacturer’s protocol. The expression levels of BOK, or Mcl-1, and 18S (housekeeping gene) were analyzed using SYBR Green Master mix (Qiagen Valencia, CA) and gene-specific primers on Applied Biosystems (ABI) Thermocycler 7900 Fast. The real-time PCR was done in triplicate for each run. Reverse transcription was performed using the iScript Reverse Transcription kit (Bio-rad Hercules, CA). Real-time PCR was conducted on an ABI Prism 7900 Fast Sequence Detection System (ThermoFisher Scientific Inc., Foster City, CA) using 95°C denaturation for 15 minutes followed by 40 cycles of 94°C for 15 seconds, then 60°C for 30 seconds, and 72 °C for 30 second. Fold change was generated using the equation 2^−ΔΔCt.