Cfx connect real time pcr detection system instrument

Isolation of total RNA was carried out using NucleoSpin RNAII (#740955, Macherey-Nagel, Duren, Germany), and the first-strand cDNA was then generated using the PrimeScript RT reagent kit (#RR037A, TaKaRa, Dalian, China). The qRT-PCR was performed using a SYBR Premix Ex Taq II kit (#RR820L, TaKaRa), according to the manufacturer's instructions. The primer sequences are listed in Table S2. Data were collected and analyzed using a CFX Connect Real-Time PCR Detection System instrument (Bio-Rad, Hercules, CA, USA).

Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany) and first-strand cDNA was synthesized from 2 µg of RNA using the RevertAid First Strand cDNA Synthesis Kit and random hexamers according to the manufacturer’s instructions (Fermentas, Burlington, Canada). First-strand cDNA from transfected CHO cells was employed as the template in real-time polymerase chain reaction (PCR) amplifications using pairs of oligonucleotide primers specific for the human Nav1.5 and β1 subunits and amplification conditions as described elsewhere^{8 (link), 55 (link)}. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as a reference gene. Sequence of the PCR-primers are detailed in the Supplementary Information. Changes in cDNA amount were quantified by real-time PCR (CFX Connect™ Real-Time PCR Detection System instrument, Bio-Rad Laboratories, Hercules, CA, USA) by using the comparative Ct method. Each sample was run in triplicate.