Sybr premix taq quantitative pcr system

Total RNA was extracted from stem cells or homogenized tissues using
illustra^TM RNAspin mini kit (GE Healthcare, Amersham, UK) and the cDNA was
generated from 2 μg of total RNA using the SuperScript^TM First-Strand Synthesis
System (Invitrogen, Carlsbad, CA, USA). Synthesized cDNAs were subjected to quantitative
real-time PCR using the Takara SYBR premix Taq quantitative PCR system (Takara Bio Inc.,
Shiga, Japan) and the MyiQ2 real-time PCR machine (Bio-Rad, Hercules, CA, USA). Primer
information and reaction conditions used for our quantitative real-time PCR analysis are
listed in Supplementary Table 1. All quantitative real-time PCR procedures, including the
design of primers, the validation of PCR conditions, and the quantification methods were
performed according to the MIQE guidelines^{17 (link)}.

Total RNA of each rat was extracted from the liver sample or cell lysate by an using illustra™ RNAspin mini kit (GE healthcare, UK). The preparation of the first-strand cDNA was conducted following the instruction of the SuperScript™ First-Strand Synthesis System (Invitrogen, Calsbad, CA). The mRNA expression levels of target genes were measured by a Takara SYBR premix Taq quantitative PCR system (Takara Bio Inc, Shiga, Japan) using a MyiQ2 real-time PCR machine (Bio-Rad). The primer sequences and annealing temperatures used in these real-time PCR reactions are listed in Suppl. Table S1. 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 a percentage of the control value in the Figures. All real-time PCR procedures including the design of primers, validation of PCR environment and quantification methods were performed according the MIQE guideline23 (link).