Iscript cdna reverse transcriptase

RNA extraction, cDNA synthesis, and real-time reverse transcriptase-polymerase chain reaction (qPCR) were performed as described by Bahamonde et al. [50 (link)]. The RNA integrity of fall 2010 samples was reported by Bahamonde et al. [50 (link)]. The RNA integrity of fall 2011 and 2012 samples was evaluated using A260/A280 and the Tape Station (2200, Agilent Technologies) with the RNA Screen Tape according to the manufacturer’s protocol. RNA Integrity Number equivalent (RIN^e) values averaged 8.93 ± 0.07 (mean ± SE). Briefly, 1 μg total RNA was reverse transcribed to cDNA using iScript cDNA reverse transcriptase (Bio-Rad Laboratories, Mississauga, ON, Canada) according to the manufacturer’s protocols. Gene-specific primer sequences for rainbow darter, vitellogenin (vtg), 18S ribosomal RNA (18s), elongation factor 1-α (ef1α), and β-actin were obtained from Bahamonde et al. [50 (link)] and are listed in Table 2. Reference genes were combined using geNORM [54 (link)], and normalized expression levels for target genes (vtg) were extracted using CFX Manager 2.1 software (Bio-Rad Laboratories) using a relative ΔΔCq method. Expression data are reported as fold change ± SE from reference (R) male mRNA levels.

Real-time PCR was used to determine levels of placental and renal PPET-1 (preproendothelin-1) as previously described [12 (link)]. Total RNA from the placenta or renal cortex was extracted using the RNeasy Protect Mini kit (Qiagen, Hilden, Germany). cDNA was synthesized from 1 μg of RNA with Bio-Rad iScript cDNA reverse transcriptase, and real-time PCR was performed using the Bio-Rad SYBR Green supermix (Bio-Rad, Hercules, CA). PPET-1 expression is calculated by ΔΔCT method using B actin as the house keeping gene and expressed as the fold difference from NP rats.