2.6. Analysis of Adipoq expression by RT‐qPCR

Visceral and subcutaneous pre‐adipocytes were incubated with CCK‐8 (10⁻⁷–10⁻⁵ M) for 2 hr, then washed with PBS, and preserved with Trizol (n = 5; samples were run in duplicate). Total RNA was extracted by using the Tri‐Reagent protocol (Life Technologies). cDNA was then synthesized from 1‐μg total mRNA by using a high‐capacity cDNA RT kit (Bio‐Rad). Quantitative RT‐PCR was performed by using designed primer pairs (Integrated DNA Technologies, USA; see Table 1). SsoAdvanced Universal SYBR Green Supermix (Bio‐Rad) was used for amplification according to the manufacturer's protocols in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, USA). All gene values were normalized to the housekeeping gene 18s and β‐Actin. The ∆∆C(T) method was used to determine relative expression levels of rat genes. Statistics were performed using the ∆∆C(T) method (Livak & Schmittgen, 2001).

Designed primer pairs used in this study