不同果糖喂养状态下大鼠肝脏的内参基因稳定性分析

发布时间：2018-04-20 00:08

  本文选题：Real-time + PCR　； 参考：《基因组学与应用生物学》2017年08期

【摘要】：分析取材前不同果糖喂养(禁食给清水和禁食给果糖水)条件下,大鼠肝脏常用内参基因m RNA的稳定性,筛选出最适宜的内参,以保证实时荧光定量PCR(real-time PCR)结果的可靠性。长期给予10%果糖水建立非酒精性脂肪肝大鼠模型,在动物体重与饮用果糖量相等的情况下,取材前随机分成两组:禁食给清水组(Fru 1);禁食继续给果糖水组(Fru 2);正常对照组(Con)自由饮清水。提取肝脏样本RNA,取等量RNA逆转录合成c DNA,real-time PCR检测18S、GAPDH、ACTB和TBP含量变化,分别利用ge Norm程序和Bio-Rad CFX Manager软件分析这几个基因的稳定性。ge Norm分析得到的稳定性排序为:ACTBGAPDHTBP18S。Bio-Rad CFX Manager软件稳定性排序为:ACTBTBPGAPDH18S。两个软件均得到ACTB最为稳定,TBP次之。然而经3次重复逆转录基因表达分析,相对于Con组和Fru1组,Fru 2组ACTB与GAPDH明显升高,18S略有下降,TBP在3组间比较稳定。结合肝脏脂质合成相关的两个关键基因Ch REBP,SREBP1c表达变化分析,分别以这4种基因为内参计算这两个基因在3组间的含量变化。只有以GAPDH为内参时,Fru 2组Ch REBP表达量相对于Fru 1没有明显上升,其它的表达趋势一致,只是含量高低有所区别。因此,我们认为TBP和ACTB均可作为不同果糖喂养状态下大鼠肝脏的内参基因。
[Abstract]:Under the condition of different fructose feeding (fasting water and fructose water) before sampling, the stability of the commonly used internal reference gene m RNA in rat liver was analyzed, and the most suitable internal parameters were selected to ensure the reliability of the results of real-time fluorescence quantitative PCR(real-time. The rat model of non-alcoholic fatty liver was established by long-term administration of 10% fructose water. When the weight of the animal was equal to the amount of fructose consumed, Before sampling, they were randomly divided into two groups: fasting group with Fru _ (1); fasting group with fructose water group (Fru _ (2)); normal control group (Conon) with free drink of clear water. RNA was extracted from liver samples, and the content of ACTB and TBP were detected by real-time PCR of RNA reverse transcriptase synthesis. The stability of these genes was analyzed by ge Norm program and Bio-Rad CFX Manager software. Ge Norm analysis showed that the stability order of these genes was:: ACTBGAPDHTBP18S.Bio-Rad CFX Manager software:: ACTBTBPGAPDH18S. ACTB is the most stable one in both software. However, compared with Con group and Fru1 group, the expression of ACTB and GAPDH in ACTB and GAPDH increased significantly after three times repeated reverse transcriptional gene analysis. Combined with the analysis of the changes of the expression of two key genes related to liver lipid synthesis, the contents of the two genes in the three groups were calculated by using these four bases. The expression of Ch REBP in Fru 2 group was not significantly higher than that in Fru 1 group with GAPDH as the internal reference. The other expression trends were the same, but the content of Ch REBP was different from that in Fru 2 group. Therefore, we think that both TBP and ACTB can be used as internal reference genes of rat liver under different fructose feeding conditions.
【作者单位】： 重庆医科大学基础医学院;中医药防治代谢性疾病重庆市重点实验室;重庆医科大学第二附属医院;
【基金】：国家自然科学基金(81374033,81673659) 重庆市教委课题(KJ1600233) 重庆市渝中区科委课题(20120220) 重庆医科大学培育基金(X12100)共同资助
【分类号】：R575.5
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本文编号：1775343