Sybr green pcr master mix detection kit

Total RNA was extracted from placental tissues (PE and normotensive control, 100 mg of tissue was homogenized) and CTB after 24 h using TRI reagent (Invitrogen, Oxford, UK). Then, 1 μg of total RNA was reverse transcribed to cDNA using the GoScript™ Reverse Transcriptase System (Promega, Madison, Wi, USA), according to the manufacturer´s instructions. Quantitative reverse transcription-PCR (qRT-PCR) was performed, as previously described [27 (link)]. In brief, qRT-PCR was carried out on a CFX qRT-PCR System using the SYBR^® Green PCR master mix detection kit (Promega, Madison, Wi, USA). The primer pairs are listed in Table 1. The relative gene expression was calculated using the 2^−ΔΔCq method, using Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase (YWHAZ) as the reference gene.

Total RNA was isolated from primary human trophoblast cells (CTB and STB stage) using Trizol reagent (Invitrogen, United Kingdom). RNA concentration and purity were determined spectrophotometrically using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, United States). Subsequently, RNA was reverse transcribed to cDNA with the GoScript™ Reverse Transcriptase System (Promega, United States) according to the manufacturer’s instructions. PCR analysis was carried out using the ViiA™7 RT-PCR System (Applied Biosystems, United States) and the SYBR^® Green PCR master mix detection kit (Promega, United States), as previously described (Kallol et al., 2018b (link)). The primers used are listed in Supplementary Table S1. The mRNA expression of target genes was normalized against the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), stably expressed during trophoblast differentiation as previously described (Kallol et al., 2018b (link)). Subsequently, relative expression to the undifferentiated cells was calculated and the results are expressed as 2^−ΔΔCt or as median log₂ Fold Change (FC).

Total RNA was extracted both from placental tissue (approximately 100 mg obtained from PE and normotensive controls) and trophoblast cells after hypoxia/reperfusion using TRI reagent (Invitrogen, UK). Total RNA (1 μg) was reverse transcribed to cDNA using the GoScript™ Reverse Transcriptase System (Promega, USA) according to the manufacturer's instructions. Quantitative reverse transcription‐PCR (qRT‐PCR) was performed as previously described.
⁴³ (link) In brief, qRT‐PCR was carried out on a CFX qRT‐PCR System using a SYBR® Green PCR master mix detection kit (Promega, USA). Primer pairs are listed in Table 1. The relative gene expression was calculated using the 2^−ΔΔCq method, using tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase (YWHAZ) as the reference gene.