Fresh placental tissue was collected within 1 h after delivery. Four standardized biopsies were taken at fixed locations across the middle point of the placenta at around 4 cm distance from the umbilical cord, on the foetal side as detailed by Janssen and colleagues [62 ,63 (link)]. The freshly collected biopsies were stored in RNA later (ThermoFisher Scientific, Massachusetts, USA) at 4°C for at least 12 hours, and maximally 1 week, after which the biopsies were stored at −20°C until extraction.
Total RNA and miRNA were extracted from a single fresh placental biopsy using the miRNeasy mini kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s protocol. RNase-Free DNase treatment was performed on RNA samples according to the manufacturer’s instructions (Qiagen, Venlo, The Netherlands). RNA quantity and sample purity were assessed by spectrophotometry (Nanodrop 1000, Isogen, Life Science, Belgium) and RNA integrity by Agilent 2100 Bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands). All RIN (RNA integrity number) values were above 6, as required for good quality microarray-based analysis [64 (link),65 (link)].
Total RNA and miRNA were extracted from a single fresh placental biopsy using the miRNeasy mini kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s protocol. RNase-Free DNase treatment was performed on RNA samples according to the manufacturer’s instructions (Qiagen, Venlo, The Netherlands). RNA quantity and sample purity were assessed by spectrophotometry (Nanodrop 1000, Isogen, Life Science, Belgium) and RNA integrity by Agilent 2100 Bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands). All RIN (RNA integrity number) values were above 6, as required for good quality microarray-based analysis [64 (link),65 (link)].
