Gotaq qpcr master mix fluorescence quantification system

RNA was extracted using an SV Total RNA Isolation System Kit (Promega) according to the manufacturer's instructions. cDNA was synthesized via reverse transcription (High Capacity Kit, Applied Biosystems). Real-time PCR for quantitative mRNA expression analyses was performed on a StepOne Plus Real-Time PCR System (Applied Biosystems) using a GoTaq qPCR Master Mix fluorescence quantification system (Promega) and the primers listed in Table S1. The standard PCR conditions were as follows: 50°C for 2 min; 95°C for 2 min; and 40 cycles of 15 s at 95°C, 30 s at 58°C, and 30 s at 72°C, followed by a standard denaturation curve. The mean threshold cycle (Ct) values were used to calculate the expression of the target gene, which was normalized to the housekeeping gene HPRT (hypoxanthine phosphoribosyltransferase) that allows for quantitative gene expression analysis using the formula 2^−ΔΔCt.

RNA was extracted using a Promega Kit (Promega) according to the manufacturer’s instructions. cDNA was synthesized via a reverse transcription (Kit High Capacity, Applied Biosystems). Real-time PCR for quantitative mRNA expression analyses was performed on a StepOne Plus Real-Time PCR System (Applied Biosystems) using a goTaq qPCR Master Mix fluorescence quantification system (Promega) and the primer sequences used as follows: RPL13a sense, 5’-GGAGGAGAAACGGAAGGA AAAG-3’, and antisense, 5’-TTTCCGTAACCTCAAGATCTGCTT-3’; NOS-2 sense, 5’-CGAAACGCTTCACTTCCAA-3’; and antisense, 5’-TGAGCCTATATTGCTGTGGCT-3’. Prdm1 sense, 5’- GACGGGGGTACTTCTGTTCA-3’; and antisense, 5’- GGCATTCTTGGGAACTGTGT. The standard PCR conditions were as follows: 50°C for 2 min; 95°C for 2 min; and 40 cycles of 15 seconds at 95°C, 30 seconds at 58°C and 30 seconds at 72°C, followed by a standard denaturation curve. The samples were normalized to RPL13a and the analyses performed using the cycle threshold (Ct) method, which allows for quantitative expression analysis using the formula 2^-ΔΔCt.