Sybr greener

HO-2 and Cse mRNA levels in the carotid bodies were analyzed by RT-qPCR with SYBR GreenER (#11764-100, Invitrogen) as previously described (20 (link)). Briefly, RNA was extracted from rat carotid bodies (two carotid bodies from a single rat) using TRIzol (Thermo Fisher) and was reverse transcribed using SuperScript III reverse transcriptase (Thermo Fisher). Relative mRNA quantification, expressed as fold change (F) was calculated using the formula = 2^{−Δ ΔCt} where ΔC_T is the difference between the threshold cycles of the given target cDNA and 18S rRNA, and Δ (ΔC_T) is the difference between the ΔC_T values under normoxia and intermittent hypoxia. PCR specificity was confirmed by omitting the template and by performing a standard melting curve analysis. The nucleotide sequences of primers used for qPCR are as follows: rat CSE: 5’-AGC GAT CAC ACC ACA GAC CAA-3’ and 5’-ATC AGC ACC CAG AGC CAA AGG-3’; rat HO-2: 5’-ACT GGG AGG AGC AGG TGA AG-3’ and 5’-GGT AGA ACT GGG TCC CTT CCC-3’; 18S rRNA: 5’-GTA ACC CGT TGA ACC CCA TT-3’ and 5’-CCA TCC AATCGG TAG TAG CG-3’.

Total RNA was reverse-transcribed using reverse transcription system (Promega). Real time PCR was performed using SYBR GreenER (Invitrogen) on the Bio-Rad PCR instrument. PCR reaction was performed according to the standard protocol with the 50°C pre-incubation for 2 min and 95°C incubation for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1min. GAPDH was included as an endogenous control. All real-time PCR reactions were performed in triplicate, and relative quantifications (RQs) were calculated using the ΔΔCt method (95% CI). All primer sets were subjected to a dissociation curve analysis and produced single peaks on a derivative plot of raw fluorescence.