Cfx connect real time rt pcr system

Real-Time PCR was performed with the CFX Connect Real-Time RT-PCR System (Bio-Rad, Hercules, CA, USA) using the SsoAdvancedTM Universal SYBR^® Green Supermix (Bio-Rad, Hercules, CA, USA) as dye. Primers were designed with NCBI’s Primer-BLAST tool, and they are reported in Table S2. Gene expression was calculated using the 2^−ΔΔCt method, and 18S was used as an endogenous control. Data were expressed as mean ± SEM. The statistical analysis was performed with Student’s t-test. Prism 7 software (GraphPad Software Inc., La Jolla, CA, USA) was used, assuming a p-value less than 0.05 as the limit of significance.

RNA from PAXgene blood RNA tubes of patient 3 and wild-type GLUT1 was extracted with PAXgene blood RNA kit (Qiagen, Hilden, Germany).
Reverse transcription was performed using the iScript cDNA synthesis Kit (BioRad, Hercules, CA, USA) and the SsoAdvanced^TM Universal SYBR^® Green Supermix (Bio-Rad, Hercules, CA, USA) was used as dye to perform qPCR on the CFX Connect Real-Time RT-PCR System (Bio-Rad, Hercules, CA, USA), using the following GLUT1 primers F: 5′-TGGCTCCTTCTCTGTGGGCCTT-3′, R: 5′-GGACACGAAGGCCAGCAGGTTC-3′. Gene expression profiling was calculated using the ΔΔCt method, and GAPDH (NM_002046) was used as housekeeping gene for normalization (F: 5′-CTTTTGCGTCGCCAG-3′, R: 5′-TTGATGGCAACAATATCCAC-3′).
cDNAs were amplified and sequenced as previously described for DNA samples, using the following primers: F: 5′-TCGGAGTCAGAGTCGCAGTG-3′, R: 5′-CAGAGAAGGAGCCAATCATGC-3′, designed respectively on 5′UTR (forward) and on exon 3 (reverse). Electropherograms were analysed with the ChromasPro software (TechnelysiumPty Ltd., Tewantin, QLD, Australia) using the wild-type GLUT1 cDNA sequence (NM_006516) as reference.