Fam labeled taqman universal pcr mastermix

RNA was extracted from primary trophoblast and HUVECs using an RNeasy mini kit (Qiagen, Valencia, CA) and quantified using the Nanodrop ND 1000 spectrophotometer (NanoDrop technologies Inc, Wilmington, DE). 0.2 μg of RNA was converted to cDNA using Applied Biosystems high capacity cDNA reverse transcriptase kit (Life Technologies) as per manufacturer guidelines.
Gene expression of VCAM-1, ET-1, MMP 14, HO-1, Endoglin, TIMP3, NQO1, GCLC, TXN, YWHAZ and GAPDH (Life Technologies) were quantified by real time PCR (RT-PCR) on the CFX 384 (Bio-Rad, Hercules, CA) using FAM-labeled Taqman universal PCR mastermix and its specific primer/probe set (Life Technologies) with the following run conditions: 50 °C for 2 minutes; 95 °C for 10 minutes, 95 °C for 15 seconds, 60 °C for 1 minute (40 cycles). SYBR RT-PCR was carried out to assess gene expressions of sFlt-1 e15a and sFlt-1 i13, YWHAZ and GAPDH. Primers were designed as previously described (Geneworks, South Australia, Australia)^{21 (link)}. RT-PCR was performed using the following run conditions: 95 °C for 20 minutes; 95 °C for 0.01 minutes, 60 °C for 20 minutes, 95 °C for 1 minute (39 cycles), melt curve 65 °C to 95 °C at 0.05 °C increments at 0.05 seconds.

qPCR analysis was conducted on mRNA extracted from preterm control, preeclampsia + IUGR and IUGR placentas. Extraction of RNA from placental cytotrophoblasts and explants were performed with the RNAeasy mini kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions and quantified using the Nanodrop ND 1000 spectrophotometer (NanoDrop technologies Inc., Wilmington, DE, USA). RNA (0.2 μg) was converted to cDNA using the Applied Biosystems high-capacity cDNA reverse transcriptase kit (Life Technologies, Carlsbad, CA, USA) in line with the manufacturer’s guidelines. We assessed gene expressions of SLC38A1 (Hs01562175_m1), SLC38A2 (Hs01089954_m1), and SLC38A4 (Hs00394339_m1) (Taqman probes, Life Technologies) by real time PCR (RT-PCR) on the CFX 384 (Bio-Rad, Hercules, CA) using FAM-labeled Taqman universal PCR mastermix and its specific primer/probe set (Life Technologies) with the following run conditions: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min (40 cycles). Quantification was performed using the 2^–∆Ct method, normalising expression to the average expression of housekeeper genes CYC1 (Hs00357717_m1) and TOP1 (Hs00243257_m1). Results are presented as mRNA expression (2^–∆Ct).