Myometrial Gene Expression Profiling

Myometrial tissues were crushed into a fine powder on dry ice. Total RNA was extracted using Trizol and further treated with DNase I to remove genomic DNA. RNA was reverse transcribed using the high-capacity cDNA synthesis kit (Thermo Fisher Scientific). Target gene expression was monitored by qPCR using SYBR Select (Thermo Fisher Scientific) and primers that target sequences within the exons of pertinent mRNA transcripts (S1, S2 Tables inS1 File), and normalized to levels of total H1f0 (Bl6 mice), Tbp (CD-1 mice) or Mapk1 (human) mRNA. These reference genes have been previously used because of their most consistent profiles of expression at similar levels across gestational time points [8 , 19 (link)]. Relative expression levels were calculated against genomic DNA-based standard curve references. All samples were confirmed not to have DNA contamination because no target amplification was observed with DNA polymerase in the qPCR reactions for reverse transcriptase-negative samples.