Biomark high throughput qrt pcr system

Dams were injected with αCD3ε, LPS, or RU486 (or their respective controls) (n = 7 – 9 each for αCD3ε, 9 – 13 each for LPS, and 11 – 16 each for RU486). Mice were euthanized 12–16 h post-injection and decidual and myometrial tissues from the implantation sites were collected and placed in RNAlater Stabilization Solution (Life Technologies). Total RNA was isolated from decidual and myometrial tissues using the RNeasy mini kit (Qiagen), following the manufacturer’s instructions. RNA concentrations and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and RNA integrity was evaluated with the 2100 Bioanalyzer system (Agilent Technologies) using the Agilent RNA 6000 Nano Kit (Agilent Technologies). Complementary (c)DNA was synthesized by using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Life Technologies) on the Applied Biosystems GeneAmp PCR System 9700 (Applied Biosystems, Life Technologies), following the manufacturer’s instructions. Complementary DNA was amplified using the TaqMan® PreAmp Master Mix (2X) (Applied Biosystems) on the Applied Biosystems 7500 Fast Real-time PCR System. Messenger RNA expression was determined by quantitative real-time PCR (qRT-PCR) using a BioMark high-throughput qRT-PCR System (Fluidigm) and TaqMan® gene expression assays (Thermo Fisher) (Supplementary Table I).

RNA was extracted from decidual and myometrial tissues using TRIzol Reagent (Life Technologies), QIAshredders (Qiagen, Valencia, CA, USA), RNase-Free DNase Sets (Qiagen), and RNeasy Mini Kits (Qiagen). RNA concentrations and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and RNA integrity was evaluated with the Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE, USA). cDNA was synthesized using RT² First Strand Kits (Qiagen). The RT² Profiler Mouse PPAR Targets PCR Array (Qiagen) and RT² Profiler Mouse Inflammatory Cytokines & Receptors PCR Array (Qiagen) were used for initial screening (n=4 samples per group) and performed by using RT² SYBR Green ROX qPCR Mastermix (Qiagen) on a 7500 Fast Real-Time PCR System (Applied Biosystems, Life Technologies Corporation, Foster City, CA, USA). Expression profiling of those genes selected based on the screening results was confirmed by qRT-PCR using a BioMark High-throughput qRT-PCR System (Fluidigm, San Francisco, CA, USA) and an ABI 7500 Fast Real-Time PCR System (Applied Biosystems) using TaqMan gene expression assays (Applied Biosystems) (n=6–8 mice per group; Supplementary Table II).