Myiq2 real time pcr machine

Total RNA of cells was extracted by using illustra^TM RNAspin mini kit (GE healthcare, UK). The preparation of the first-strand cDNA was conducted following the instruction of the SuperScript^TM First-Strand Synthesis System (Invitrogen, Calsbad, CA). The mRNA expression levels of Bcl-2, Bax1, NAD(P)H:quinone oxidoreductase-1 (NQO-1), malic enzyme-1 (ME-1), oncostatin M (OSM) and epidermal growth factor (EGF) (for sequence information, see Supplementary Table 1) were measured by Takara SYBR premix Taq quantitative PCR system (Takara Bio Inc, Shiga, Japan) and in MyiQ2 real-time PCR machine (Bio-Rad, Hercules, CA). Parallel amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Relative quantification was done by using the 2^−ΔΔCt method. The relative expression of the specific gene to the internal control was obtained and then expressed as percentage of the control value. All real-time PCR procedures including the design of primers, validation of PCR environment and quantification methods were performed according the MIQE guideline18 (link).

Total RNA was extracted using illustraTM RNAspin mini kit (GE Healthcare, UK) and then reverse-transcribed using SuperScriptTM First-Strand Synthesis System (Invitrogen, Calsbad, CA, USA). The mRNA expression levels were measured using Takara SYBR premix Taq quantitative PCR system (Takara Bio Inc, Shiga, Japan) and MyiQ2 real-time PCR machine (Bio-Rad). The sequences for the specific primers are listed in Supplementary Table 1. Parallel amplification of GAPDH was used as the internal control. Relative quantification was done by using the 2^−△△Ct method. The relative expression of the specific gene to the internal control was obtained and then expressed as a percentage of the control value in the figures. All quantitative PCR procedures including the design of primers, validation of PCR environment and quantification methods were performed according the MIQE guideline11 (link)12 (link).