Applied biosystems 7900ht instrument

Real-time RT-PCR (q-PCR) analysis was performed using the SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies) and an Applied Biosystems 7900HT instrument (Applied Biosystems, Foster, CA). The specific primers used are listed in the Supplementary Table S1. All quantitative real-time RT-PCRs were performed in four replicates. Gene regulation was quantitated by the 2^−ΔΔCt method with normalization to the GAPDH level.

Total RNA was extracted from uterus, fetal membranes, and placental tissue collected 6 h postsurgery using the RNeasy mini kit (Qiagen, Crawley, U.K.) as per the manufacturer’s guidelines. Total RNA (300 ng) was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Life Technologies). qRT-PCR was carried out to quantify the mRNA expression of specific genes of interest. Predesigned gene expression assays from Applied Biosystems were used to examine the expression of Cxcl1 (Mm04207460_m1), Cxcl2 (Mm00436450_m1), Cxcl5 (Mm00436451_g1), Il-1β (Mm00434228_m1), the neutrophil marker neutrophil granule protein (Ngp; Mm01250218_m1), and Tnf-α (Mm99999068_m1). Primers and probes for β-actin and Il-6 were designed using Primer Express Software Version 3 (Applied Biosystems). Details of designed β-actin and Il-6 primer and probe sequences are given in Table I. Target gene expression was normalized for RNA loading using β-actin, and the relative expression in each sample was calculated relative to a calibrator sample (untreated day 18 uterus), which was included in all reactions, using the 2^−ΔΔ threshold cycle method of analysis. All qRT-PCR analysis was performed on an Applied Biosystems 7900HT instrument (Applied Biosystems).

Total RNA was extracted using TRIzol (Invitrogen Inc., Carlsbad, CA, USA) from RASFs or OASFs that were incubated with different doses of AD, and the RNA was reverse-transcribed using the PrimeScript RT-PCR Kit according to the manufacturer’s instructions (TaKaRa). Quantitative real-time PCR was conducted using the Applied Biosystems 7900HT Instrument (Applied Biosystems, Carlsbad, CA, USA) and the SYBR Green PCR Master Mix. The primer sequences for the genes were as follows: OPN, sense 5’-GAAGTTTCGCAGACCTGACAT-3’, antisense 5’-GTATGCACCATTCAACTCCTCG-3’; GAPDH, sense 5’-TGACTTCAACAGCGACACCCA-3’, antisense 5’-CACCCTGTTGCTGTAGCCAAA-3’. The cycling conditions included an initial denaturation procedure at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Relative gene expression was determined by 2^-ΔΔCt.