Taqman gene expression master mix

Placentas were quickly dissected after the isolation of the serum and CSF of fetuses at 12.5 dpc and 13.5 dpc. Total RNA of placentas was extracted using RNeasy (Qiagen, Valencia, CA). Expression levels of Proopiomelanocortin (Pomc), Il-6st (gp 130), Lifr (Lif receptor), Il-6r (Il-6 receptor) and Socs3 were examined using a gene expression assay (Applied Biosystems, Foster City, CA). POMC is the precursor of ACTH and melanocortin peptides. A total of 2 μg of RNA was reverse-transcribed using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). Quantitative PCR was performed using cDNA with TaqMan gene expression master mix (TakaRa Bio, Shiga, Japan), and TaqMan 18S ribosomal RNA (Applied Biosystems) as an internal control. Reactions were performed in triplicate using ABI Prism 7900HT (Applied Biosystems) and quantified using the delta–delta-cycle threshold (Ct) method (2 ^-ΔΔCt) [21 (link)]. Three or four dams for each group were used, and two fetuses were collected as a pooled sample from each litter.

Total RNA of placenta and mTSCs was extracted using a RNeasy mini kit (Qiagen, Valencia, CA, United States). Expression levels of Crh were determined using a gene expression assay (Applied Biosystems, Foster City, CA, United States). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen), and quantitative PCR was performed using cDNA with TaqMan gene expression master mix (Takara Bio, Kusatsu, Japan). Crh (Mm01293920_s1) expression was normalized to the housekeeping genes of 18S ribosomal RNA (Applied Biosystems) using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)). Pomc RT-PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus, Takara Bio) and a Thermal Cycler Dice Real-Time System III instrument (Takara Bio). Each PCR reaction consisted of 6.25 μL TB Green Premix Ex Taq II, 4.5 μL water, 1 μL cDNA, and 0.5 μL (0.39 μM) Pomc (NM_008895) gene-specific primer (Simard et al., 2010 (link)). The cycle threshold for Pomc amplification was normalized to the expression level of 18S ribosomal RNA. Reactions were performed in triplicate.

After inoculating on 2D TCP, 3D GelMA, and 100G in SCM and SFM, respectively, for 2 weeks, cells were lysed by Trizol (Invitrogen) to extract RNA. The concentrations of RNA were measured using a Nanodrop 2000 (Thermo scientific). The same amount of RNA was transcribed to cDNA by using a Quantitative Reverse Transcription kit (Takara) according to the manufacturer’s protocol. TaqMan gene expression master mix (Takara) and the primers for the studied gene (Supplementary, Table 1) were added to sample cDNA (2 μL), and a StepOnePlus real time PCR system (Applied Biosystems) instrument was used with 40 amplification cycles. Cells cultured on 2D TCP were used as a reference for comparison. β-actin was utilized as an endogenous control for the normalization of studied genes expression levels using the delta delta Ct (ΔΔCt) method. Calculation of 2^{−ΔΔ Ct} was performed to give a comparative fold change of gene expression level relative to actin. Three replicates were measured for each sample to calculate the mean and standard deviation.