Perfecta sybr green supermix low rox

qPCR analyses were performed in 96-well plates on a ViiA7 Real-time PCR machine (Thermo Fisher Scientific) and the reactions contained 2 μl of cDNA template, 5 μl of PerfeCTa SYBR green Supermix Low ROX (Thermo Fisher Scientific), 0.25 μM for each forward and reverse primer targeting RG candidates (Table 1), and nuclease free water in a total volume of 10 μL. qPCR reactions were initiated by denaturation at 95 °C for 20 s, followed by 40 cycles of amplification. The thermal cycling profile consisted of denaturation at 95 °C for 1 s and annealing and extension at 60 °C for 2 s with subsequent acquisition of fluorescence data. A melting curve was generated (95 °C for 15 s, 65 °C for 1 min, 95 °C for 15 s) to discriminate between specific and non-specific amplification products (in all cases the ramp time was 1 °C/s). All qPCR reactions were run in duplicate; amplification efficiencies were calculated for each primer pair by standard curves using 8 points of 10-fold dilution series from standards obtained for each candidate gene ranging from 10⁸ to 10¹ copies/µL (Table 1).

LPA receptor mRNA levels in tissues and cells were determined using qPCR using validated optimal reference gene pairs as previously described [28 (link)]. Primer information for LPA receptor and reference genes is provided in Table 2. Ground tissue samples and harvested cells were homogenized in Ribozol (Amresco), RNA was isolated as per the manufacturer’s instructions, and RNA quality and quantity were examined using a QIAxcel Advanced System (Qiagen). cDNA was synthesized from 1 μg of RNA using qScript cDNA supermix (Quanta Biosciences) and cDNA samples were stored at −20°C until further use. qPCR analysis was performed in 96-well plates using PerfeCTa SYBR green Supermix Low ROX (Thermo Fisher Scientific) and a ViiA7 real-time PCR machine (Thermo Fisher Scientific) as detailed previously [28 (link)]. mRNA levels were quantified using gbase + software (Biogazelle) [28 (link)].