RNA extraction and Real Time PCR analysis
Total RNA from all left and right OB tissue was extracted using the TRIzol™ reagent, according to the manufacturer specifications (Invitrogen-Life Technologies, Buenos Aires, Argentina.). mRNA integrity samples were confirmed by 1% agarose gel electrophoresis and staining with Sybr Gold™ (Invitrogen-Life Technologies, Buenos Aires, Argentina). 10 μg
The mRNA levels of TPH2 and GABAA α1 were estimated by RT real-time PCR with a Corbett Rotor-Gene 6000 Real-Time Thermocycler (Corbett Research Pty Ltd (Sydney, Australia) using rat-specific primers and reaction conditions described in Table 1. The PCR reactions were performed using a Corbett Rotor-Gene 6000 Real-Time Thermocycler using Eva-GreenTM (Biotium, Hayward, CA) in a final volume of 20 μL. The reaction mixture consisted of 2 μL of 10×PCR Buffer, 1 μL of 50 mM MgCl2, 0.4 μL of 10 mM dNTP Mix (Invitrogen), 1 μL of 20× Eva Green, 0.25 μL of 5 U/μL Taq DNA Polymerase (Invitrogen) 0.1 μL of each 2.5 mM primer (forward and reverse primers) and 10 μL of diluted cDNA. The PCR reactions were performed under the conditions described in Table 1. Melt curve analysis was used to check that a single specific amplified product was generated. Real-time quantification was monitored by measuring the increase in fluorescence caused by the binding of EvaGreen dye to double-strand DNA at the end of each amplification cycle. According to the manufacturer protocol, the relative expression was determined using the Comparative Quantitation method of normalized samples about the expression of a calibrator sample (Pfaffl, 2001). Each PCR run included a no-template control and a sample without reverse transcriptase. All measurements were performed in duplicate. The reaction conditions and quantities of cDNA added were calibrated such that the assay response was linear concerning the amount of input cDNA for each pair of primers. RNA samples were assayed for DNA contamination by performing the different PCR reactions without prior reverse transcription. Relative levels of mRNA were normalized to the S16 reference gene. The real-time PCR products were analyzed on 2% agarose gels containing 0.5 mg/mL ethidium bromide and a unique band of the approximately correct molecular weight corresponded with a unique peak in melt curve analysis.
The Real-Time PCR reactions were carried out for 40 cycles with an initial step of 5 min at 95 ºC followed by a three-step scheme: 30 s at 95 ºC, 30 s at the annealing temperature shown above for each primer pair, and a final step at 72 ºC for 30 s. Primer’s design was done with Beacon Designer 7.9 software.