Mastercycler ep realplex4s real time pcr system

Total RNA was extracted from liver subcutaneous adipose and visceral adipose tissues using Trizol reagent (TaKaRa, Otsu, Japan). First-strand complementary DNA (cDNA) was generated by reverse transcriptase, with random primers (TaKaRa, Otsu, Japan). The sequences of the primers are described in Table 1. To evaluate the mRNA expression of CDKAL1 in the liver subcutaneous adipose and visceral adipose tissues, real-time PCR was performed using a SYBR Green master mix kit (TaKaRa, Otsu, Japan) according to the manufacturer's instructions on the Mastercycler ep realplex4S real-time PCR system (Eppendorf, Hamburg, Germany). The cDNA was denatured at 95°C for 10 min followed by 40 cycles of PCR (95°C, 15 s; 60°C, 60 s). The 2^−ΔΔCt method [15 (link)] was used to determine relative amounts of product, and data are presented as fold change, using β-actin as an endogenous control.

Total RNA was extracted from liver tissues and visceral fat using RNAiso plus (TaKaRa, Otsu, Japan). Complementary DNA (cDNA) was synthesized using random primers and reverse transcriptase as recommended by the manufacturer instructions (TaKaRa, Otsu, Japan). To evaluate the mRNA expression of lipid transport factors that were derived from caveolae in liver tissues and visceral fat, real-time PCR was conducted using a SYBR Green PCR master mix (TaKaRa, Otsu, Japan) according to the manufacturer's instructions. The reactions were carried out in the Mastercycler ep realplex 4S real-time PCR system (Eppendorf, Hamburg, Germany). The sequences of the primers are described in Table 1. The cDNA was denatured at 95°C for 30s followed by 40 cycles of PCR that included a denaturation step at 95°C for 5 s and an annealing step at 60°C for 30 s. The mRNA levels of the target genes were normalized to the mRNA levels of the β-actin gene and the results were expressed as fold changes of the threshold cycle (Ct) value relative to the control samples using the 2^−ΔΔCt method [41 (link)].