SYBR green qPCR assay

SYBR green qPCR was prepared by mixing the following reagents in each well of a MicroAmp Optical 384-well reaction plate (Applied Biosystems, 4309849): 1× Power SYBR Green PCR Master Mix (Applied Biosystems, 4367659), forward and reverse primers (4 μM, 1 μl each), cDNA template (1 μl), and nuclease-free H₂O in a total volume of 10 μl. The PCR and optical reading of the plate were performed using a QuantStudio 5 thermocycler. qPCRs were performed in triplicate for each sample. SYBR green qPCR cycle conditions were 50°C for 2 min for 1 cycle; 95°C for 10 min for 1 cycle; 95°C for 15 s, 60°C for 1 min for 40 cycles; 95°C for 15 s, 60°C for 1 min, then the temperature was slowly raised to 95°C at a rate of 0.075°C per second for melt curve determination.

The following primers were used: human Scn1a productive transcript, 5′-ATTGTTGATGTTTCATTGGTCAGTTTAACA-3′ (forward) and 5′-GGCATTCACAACCACCCTCATC-3′ (reverse); human RPL32 transcript, 5′-AGAGGCATTGACAACAGGGTT-3′ (forward) and 5′-GTGAGCGATCTCGGCACAG-3′ (reverse).