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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:ACTB>GAPDH>TBP>18S。Bio-Rad CFX Manager软件稳定性排序为:ACTB>TBP>GAPDH>18S。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。
The stability of m RNA, which is commonly used in rat liver, was analyzed under different fructose feeding conditions (fasting to fresh water and fasting to fructose-water), and the most suitable internal control was screened to ensure real-time PCR) the reliability of the results. Long-term given 10% fructose water to establish non-alcoholic fatty liver rat model, in the case of animal weight and drinking fructose equal to the circumstances, were randomly divided into two groups: fasting group (Fru 1); fasting continue fructose Water group (Fru 2); normal control group (Con) free to drink water. The RNA of liver samples was extracted and the same amount of RNA was reverse transcribed to synthesize c DNA. The contents of 18S, GAPDH, ACTB and TBP were detected by real-time PCR. The stability of these genes was analyzed by ge Norm program and Bio-Rad CFX Manager software . ge Norm analysis of the stability of the order: ACTB> GAPDH> TBP> 18S. Bio-Rad CFX Manager software stability was ranked as: ACTB> TBP> GAPDH> 18S. ACTB was the most stable of both software, followed by TBP. However, after three repeated RT-PCR analyzes, ACTB and GAPDH in Fru 2 group were significantly higher than those in Con group and Fru1 group, 18S decreased slightly, and TBP was more stable among the three groups. Combined with the analysis of changes in the expression of ChREBP and SREBP1c, two key genes involved in liver lipid synthesis, the changes of these two genes in the three groups were calculated respectively using these four genes as internal reference. Only when GAPDH was used as an internal control, the expression level of Ch REBP in Fru 2 group did not increase significantly compared with that in Fru 1 group. The other expression trends were the same, except for the difference in content. Therefore, we think both TBP and ACTB can be used as internal reference genes of rat liver under different fructose feeding conditions.