Sybre green supermix

To determine if local expression of Fas and/or FasL was altered in response to HELLP syndrome mRNA was extracted from individual liver, placenta, kidney cortex, and kidney medulla as previously described [16 ]. Briefly, individual tissues (n=4–6/group) were crushed in liquid nitrogen, and mRNA was extracted using a Qiagen kit (Venlo, Netherlands) as outlined in the manufacturer’s instructions followed by treatment with DNAse per manufacturer’s directions (Applied Biosystems, Foster City, CA). cDNA was synthesized from 1μg of RNA with BioRad Iscript cDNA reverse transcriptase kit (Hercules, CA). Real-time polymerase chain reaction was performed using BioRad Sybre Green Supermix with an iCycler. Beta actin Ct (threshold cycle) was used as a housekeeping gene and the 2^−ΔΔCt method was used to analyze the results.
delta/delta Ct method was used to assess gene expression. Primer sequences [16 , 21 (link)] were provided by Life technologies (Carlsbad, CA).

Placental total RNA was extracted using the RNeasy ® Protect Mini kit (Qiagen, Hilden, Germany). Real-time PCR was used to determine levels of placental preproendothelin-1 (PPET-1). cDNA was synthesized from 1μg of RNA with Bio-Rad Iscript cDNA reverse transcriptase, and real-time PCR was performed using the Bio-Rad Sybre Green Supermix (Bio-Rad, Hercules, CA). Placental PPE expression is measured as the fold difference from RUPP vs. NP and RUPP+’n7AAc’ vs. NP.^{16 (link),32}