Rotor gene 6000 real time analyzer

Total RNA was extracted from endometrium stem cells and 1 × 10⁶ mononuclear cells using TRIzol^® reagent (Zymo Research, Irvine, CA, USA) and Quick-DNA/RNA™ Miniprep Kit (Zymo research, Irvine, CA, USA) respectively according to manufacturer’s instructions. cDNA was synthesized with LunaScript^® RT SuperMix Kit (New England Biolabs, Ipswich, MA, USA) following manufacturers recommendations. RT-qPCR was performed using Luna^® Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) and Rotor-Gene 6000 Real-time Analyzer (Corbett Life Science, QIAGEN, Hilden, Germany) with experimental conditions as follows: 95 °C 1 min, 95 °C 15 s, 60 °C 30 s. The list of primers and their sequences used in gene expression analysis is presented in Table 3. The acquired data from RT-qPCR was further analysed, each sample’s mRNA levels were normalised to GAPDH gene expression and relative gene expression was calculated using ∆∆Ct method.

For the total RNA extraction from follicular fluid cells, the Quick-DNA/RNA Miniprep Kit (Zymo Research, CA, United States) was used in line with manufacturer’s recommendations. The RNA isolation from the endometrium tissue required an additional step prior to using the kit. A piece of the tissue was first submerged in liquid nitrogen and ground to a powder using a mortar and pestle. Then the powder was mixed well with DNA/RNA lysis buffer and the lysate was transferred to a Zymo-Spin™ Column. The following isolation steps were carried out according to manufacturer’s recommendations. After this, cDNA was synthesized with the LunaScript^® RT SuperMix Kit (New England Biolabs, Ipswich, MA, United States) following manufacturer’s guidelines. cDNA was then amplified using the RT-qPCR with Luna^® universal qPCR Master Mix (New England Biolabs, Ipswich, MA, United States) and the Rotor-Gene 6000 Real-time Analyzer (Corbett Life Science, QIAGEN, Hilden, Germany) with experimental conditions as follows: 95°C 1 min, 95°C 15 s, 60°C 30 s (40 cycles). Primer sequences used in RT-qPCR analysis are listed in Table 1. The acquired data from RT-qPCR was further analyzed using the ΔΔCt method, mRNA expression was normalized using GAPDH gene expression and therefore the relative gene expression was calculated.

RNA from differentiated and undifferentiated EndSCs and MenSCs was isolated by using TRIzol^® reagent (Zymo Research, CA, USA), chloroform and isopropanol for RNA precipitation. After this cDNA was synthesized with SensiFAST cDNA Synthesis Kit (Bioline, London, UK) by following manufacturers recommendations. RT-qPCR was performed using SensiFAST SYBR NoRox Kit (Bioline, London, UK) and Rotor-Gene 6000 Real-time Analyzer (Corbett Life Science, QIAGEN, Hilden, Germany) with experimental conditions as follows: 95°C 2 min, 95°C 5 s, X °C (primer melting temperature) 10 s, 72°C 20 s. RT-qPCR was repeated for 40 cycles for all genes and samples. Used primers are listed in the Supplementary Table S1. The acquired data from RT-qPCR was further analyzed using ∆∆Ct method. By using this method every sample was compared to undifferentiated control and normalized using GAPDH gene expression and therefore relative gene expression was calculated.