冷暴露和CL316243刺激小鼠脂肪组织内参基因的筛选

Screening of reference genes in adipose tissue with cold exposure and CL316243 stimulation in mice

[目的]筛选冷暴露和CL316243刺激时脂肪组织中稳定表达的内参基因。[方法]8~9周龄C57BL/6J雄性小鼠分成4组:冷暴露组、常温对照组、CL316243组和生理盐水对照组。将冷暴露组和常温对照组分别置于4℃和常温饲养6 h;给予CL316243组7 d皮下注射CL316243(1 mg/kg/d),生理盐水对照组注射相同体积的生理盐水。荧光定量PCR检测棕色和白色脂肪中10个内参基因的mRNA水平,采用geNorm、Normfinder、Best Keeper和Delta-Ct四种方法来评估内参基因表达的稳定性。[结果]稳定性综合排名前4的内参基因是:冷暴露时棕色脂肪中为YWHAZ、36B4、PPIA和HPRT;白色脂肪中为HPRT、YWHAZ、B2M和TBP。CL316243刺激时棕色脂肪中为PPIA、HPRT、YWHAZ和β-actin;白色脂肪中为PPIA、HPRT、TBP和18S。冷暴露和CL316243刺激时脂肪组织中配对变异值V2/3均小于0.15。[结论]冷暴露和CL316243刺激时脂肪组织中最佳内参基因的数量均为2个。冷暴露时脂肪组织最佳内参基因组合为YWHAZ和HPRT,CL316243刺激时脂肪组织最佳内参基因组合为PPIA和HPRT。

[Objective]To screen for the most stable reference genes in adipose tissue under cold exposure and CL316243 stimulation in mice.[Method]Eight to nine-week old male C57BL/6J mice were divided into the following four groups:(1)cold exposure group,(2)the control group with room temperature,(3)the CL316243 group,and(4)the saline group.The cold exposure group and the control group with room temperature were kept at 4℃and room temperature for 6 hours respectively.The CL316243 group were received intraperitoneal injection of CL316243(1 mg/kg/d)for 7 days,and the saline group were injected with the same volume of saline.The m RNA expression of 10 reference genes was measured by real-time q PCR in the brown adipose tissue and the white adipose tissue.The data were analyzed by the Ge Norm,Norm Finder,Best Keeper and Delta-Ct methods.[Result]YWHAZ,36B4,PPIA and HPRT were ranked top four in the brown adipose tissue and HPRT,YWHAZ,B2M and TBP were ranked top four in the white adipose tissue under cold exposure.PPIA,HPRT,YWHAZ andβ-actin were ranked top four in the brown adipose tissue and PPIA,HPRT,TBP and 18S were ranked top four in the white adipose tissue with CL316243 stimulation.The paired variation value of V2/3 was<0.15 in the adipose tissue with cold exposure and CL316243 stimulation.[Conclusion]The optimal number of reference genes was two in the adipose tissue with cold exposure and CL316243 stimulation.YWHAZ and HPRT were the most stable reference genes in the adipose tissue with cold exposure,whereas PPIA and HPRT were the most stable reference genes in the adipose tissue with CL316243 stimulation.