Lymphocytes separation medium

The lymphocytes separation medium (TBDscience, China) was used for the isolation of mononuclear cells from bone marrow aspirate. The Trizol reagents were added (Invitrogen, USA) to the sample and let stand for 10 minutes. Then chloroform was added, mixed well and let stand for 3 minutes. After centrifugation at 12,000 × g for 15 minutes at 4°C, the supernatant was taken and mixed with isopropyl alcohol of equal volume and let stand for 10 minutes. The supernatant was removed after centrifugation at 12,000 × g for 10 minutes at 4°C, and the precipitate was washed with ethanol. After centrifugation at 7,500 × g for 5 minutes at 4°C, the supernatant was removed and allowed to stand and dry for 15 minutes. Mix with DNase for 30 minutes and wash once with ethanol. Finally, RNase-free DDW was used to dissolve the RNA. Monitoring RNA contamination and degradation was carried out using AGAR gels. The NanoPhotometer spectrophotometer (IMPLEN, USA) was used to determine the purity of RNA, while the Bioanalyzer 2100 system (Agilent Technologies, USA) was used to determine the integrity of RNA.

Samples of placenta from patients were snipped respectively and tissue samples were snap frozen in liquid nitrogen and stored at -80°C before further processing. Umbilical cord blood samples were collected in Ethylene Diamine Tetraacetic Acid (EDTA)-treated tubes at delivery. Peripheral blood mononuclear cell (PBMCs) were isolated from 3ml anticoagulated cord blood using Lymphocytes Separation Medium (TBD science, Tianjin, China), the PBMCs were washed with phosphate-buffered saline at 4°C, the PBMCs was stored at -80°C until further test.