Mx3000p multiplex quantitative pcr system

The total RNA of A549 cells was extracted using TRIzol reagent (Takara) and then reverse-transcribed using the PrimeScript^™ RT Reagent Kit (Takara). RT-PCR amplification of the cDNA was performed using primers for the Na+/K+-ATPase α subunits and the Stratagene Mx3000P Multiplex Quantitative PCR System with SYBR Premix Ex Taq^™ (Takara). The primers used for PCR amplification of human ATP1A1 (encoding the α1 subunit), ATP1A2 (encoding the α2 subunit) and ATP1A3 (encoding the α3 subunit) were listed in S1 Table.

Total RNA was extracted from cell samples by using Trizol (Invitrogen, Carlsbad, CA, USA). Total RNA (1 μg) from each sample was treated with DNase and converted to complementary DNA (cDNA) using a PrimeScript RT Reagent kit (Takara, Dalian, China). A Stratagene Mx3000p Multiplex Quantitative PCR System with SYBR Premix DimerEraser (Takara) was used for the evaluation of mRNA levels.
Quantitative polymerase chain reaction (qPCR) was carried out at 95°C for 2 minutes, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Reactions were performed in triplicate and products were subjected to melting curve analysis and visualized on an agarose gel to confirm size. Relative gene expression levels were calculated using the 2^−ΔΔCt method [26 (link)]. Mean expression levels were represented as the ratio between the different detectors and ACTB expression. The primers for GSTP1 [27 (link)] and ACTB are listed in Table 2.