Platinum qpcr supermix udg with rox

qPCR analysis was performed as described before [22 (link),23 (link)]. Briefly, total RNA was isolated from the placenta or egWAT samples with a TriReagent^® solution (Sigma, Steinheim, Germany) and 1 µg was converted into cDNA. 2.5 µL of cDNA was used in a TaqMan assay (Platinum^® qPCR SuperMix-UDG with Rox, Invitrogen, Carlsbad, CA, USA) to detect expression levels of IL-6, CD31 and vWF in the placenta, or IL-6 in egWAT. For Tie-1, we used 1 µL of cDNA in a SYBR assay (GoTaq qPCR Master Mix, Promega, Madison, WI, USA). For normalization, HPRT or β-actin expression levels were assessed. Oligonucleotides used for the analyses are listed in Supplementary Materials, Table S2. Results were analyzed using the deltadeltaCT method, expressed as fold-induction compared to the corresponding control group. Analysis was performed on multiple placentas per dam as indicated in the figure legend.

RNA was extracted from cells using the RNeasy Plus Micro kit (Qiagen, #74004). An aliquot of total RNA was reverse transcribed using an oligo(dT) primer (Invitrogen, #18418-20). For the thermal cycle reactions, the cDNA template was amplified (Applied Biosystems Quantstudio 12K Flex Real-Time PCR System) with gene-specific primer sets using the Platinum qPCR SuperMix-UDG with ROX (Invitrogen, #11743-100) under the following reaction conditions: 40 cycles of PCR (95°C for 15 s and 60°C for 1 min) after an initial denaturation (95°C for 2 min). Fluorescence was monitored during every PCR cycle at the annealing step. mRNA levels were normalized using glyceraldehyde-3-phosphate dehydrogenase as a housekeeping gene.